Methods of therapy for B-cell malignancies using antagonist anti-CD40 antibodies

ABSTRACT

Methods of therapy for B-cell malignancies are provided. The methods comprise administering a therapeutically effective amount of an antagonist anti-CD40 antibody or antigen-binding fragment thereof to a patient in need thereof. The antagonist anti-CD40 antibody or antigen-binding fragment thereof is free of significant agonist activity when the antibody binds a CD40 antigen on a normal human B cell, exhibits antagonist activity when the antibody binds a CD40 antigen on a malignant human B cell, and can exhibit antagonist activity when the antibody binds a CD40 antigen on a normal human B cell. Antagonist activity of the anti-CD40 antibody or antigen-binding fragment thereof beneficially inhibits proliferation and/or differentiation of malignant human B cells.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application is a continuation application of U.S.application Ser. No. 12/568,373, filed Sep. 28, 2009, now U.S. Pat. No.7,820,170, which is a continuation application of U.S. application Ser.No. 12/246,029, filed Oct. 6, 2008, now U.S. Pat. No. 7,611,708, whichis a continuation application of U.S. application Ser. No. 11/849,107,filed Aug. 31, 2007, now U.S. Pat. No. 7,445,780, which is acontinuation application of U.S. application Ser. No. 10/380,223, filedMar. 11, 2003, now U.S. Pat. No. 7,288,252, which is a National Stage ofInternational Application No. PCT/US01/30963, filed Oct. 2, 2001, whichclaims the benefit of U.S. Provisional Application No. 60/237,556, filedOct. 2, 2000, the contents of each of which are herein incorporated byreference in their entirety.

FIELD OF THE INVENTION

The present invention is directed to methods of therapy for diseasescharacterized by malignant B cells and tumors of B-cell origin usingantagonist anti-CD40 antibodies or antigen-binding fragments thereof.

BACKGROUND OF THE INVENTION

B cells play an important role during the normal in vivo immuneresponse. A foreign antigen will bind to surface immunoglobulins onspecific B cells, triggering a chain of events including endocytosis,processing, presentation of processed peptides on MHC-class IImolecules, and up-regulation of the B7 antigen on the B-cell surface. Aspecific T-cell then binds to the B cell via T-cell receptor (TCR)recognition of the processed antigen presented on the MHC-class IImolecule. Stimulation through the TCR activates the T cell and initiatesT-cell cytokine production. A second signal that further activates the Tcell is an interaction between the CD28 antigen on T cells and the B7antigen on B cells. When the above-mentioned signals are received, theCD40 ligand, which is not expressed on resting human T cells, isup-regulated on the T-cell surface. Binding of the CD40 ligand to theCD40 antigen on the B-cell surface stimulates the B cell, causing the Bcell to mature into a plasma cell secreting high levels of solubleimmunoglobulin.

CD40 is a cell-surface antigen present on the surface of both normal andneoplastic human B cells, dendritic cells, monocytic and epithelialcells, some epithelial carcinomas, and on antigen presenting cells(APCs). CD40 expression on APCs plays an important co-stimulatory rolein the activation of both T helper and cytotoxic T lymphocytes. CD40receptors are also found on eosinophils, synovial membranes inrheumatoid arthritis, activated platelets, inflamed vascular endothelialcells, dermal fibroblasts, and other non-lymphoid cell types. The CD40receptor is expressed on activated T cells, activated platelets, andinflamed vascular smooth muscle cells. CD40 is also expressed at lowlevels on vascular endothelial cells and is up-regulated in areas oflocal inflammation.

Human CD40 is a peptide of 277 amino acids having a predicted molecularweight of 30,600, with a 19 amino acid secretory signal peptidecomprising predominantly hydrophobic amino acids. The CD40 receptorexists in a highly modified glycoprotein state on the cell surface andmigrates in sodium dodecyl sulfate (SDS)-polyacrylamide gels as anapproximately 50 kDa polypeptide.

The CD40 antigen is known to be related to the human nerve growth factor(NGF) receptor, tumor necrosis factor-α (TNF-α ) receptor, and Fas,suggesting that CD40 is a receptor for a ligand with important functionsin B-cell activation. During B-cell differentiation, the molecule isfirst expressed on pre-B cells and then disappears from the cell surfacewhen the B cell becomes a plasma cell. The CD40 cell-surface antigenplays an important role in B-cell proliferation and differentiation.

Binding of its ligand (termed CD40L or CD154) to the CD40 receptorstimulates B-cell proliferation and differentiation, antibodyproduction, isotype switching, and B-cell memory generation. The humanand murine CD40L (CD40 receptor) genes have been cloned (Spriggs et al.(1992) J. Exp. Med. 176:1543; Armitage et al. (1992) Nature 357:80; andU.S. Pat. No. 6,264,951). Engagement of CD40 receptors by the CD40ligand on APCs, such as macrophages and dendritic cells, up-regulatescell-surface expression of MHC Class II and CD80/86, and induces thesecretion of pro-inflammatory cytokines such as IL-8, IL-12, and TNF,all of which increase the potency of antigen presentation to T cells.

All B cells express common cell surface markers, including CD40.Transformed cells from patients with low- and high-grade B-celllymphomas, B-cell acute lymphoblastic leukemia, multiple myeloma,chronic lymphocytic leukemia, and Hodgkin's disease express CD40. CD40expression is also detected in two-thirds of acute myeloblastic leukemiacases and 50% of AIDS-related lymphomas. Further, malignant B cells fromseveral tumors of B-cell lineage express a high degree of CD40 andappear to depend on CD40 signaling for survival and proliferation.

Additionally, immunoblastic B-cell lymphomas frequently arise inimmunocompromised individuals such as allograft recipients and othersreceiving long-term immunosuppressive therapy, AIDS patients, andpatients with primary immunodeficiency syndromes such as X-linkedlymphoproliferative syndrome or Wiscott-Aldrich syndrome (Thomas et al.(1991) Adv. Cancer Res. 57:329; Straus et al. (1993) Ann. Intern. Med.118:45). These tumors appear to arise as a result of impaired T-cellcontrol of latent Epstein-Barr virus (EBV) infection. Similar lymphomasof human origin can be induced in mice with severe combinedimmunodeficiency syndrome (SCID) by inoculation of peripheral bloodlymphocytes (PBL) from healthy, EBV-positive individuals (Mosier et al.(1988) Nature 335:256; Rowe et at (1991) J. Exp. Med. 173:147).

The pathogenesis of low-grade B-lineage malignancies, includingnon-Hodgkin's lymphoma and chronic lymphocytic leukemia, is stronglyaffected by the imbalance of the growth/survival signal by CD40 and acrippled death signal by Fas. Studies in low-grade non-Hodgkin'slymphoma suggest that the disease is the result of an accumulation oflymphomatous cells due to reduction in Fas-mediated apoptosis and anincrease in the survival signal through CD40. CD40 provides a survivalsignal for lymphoma cells from non-Hodgkin's B-lymphoma patients andstimulates their growth in vitro (Romano et al. (2000) Leuk. Lymphoma36:255-262; Furman et al. (2000) J. Immunol. 164:2200-2206; Kitada etal. (1999) Br. J. Haematol. 106:995-1004; Romano et al. (1998) Blood92:990-995; Jacob et al. (1998) Leuk. Res. 22:379-382; Wang et al.(1997) Br. J. Haematol. 97:409-417; Planken et al. (1996) Leukemia10:488-493; and Greiner et al. (1997) Am J. Pathol. 150:1583-1593).

Approximately 85% of non-Hodgkin's lymphomas, a diverse group ofmalignancies, are of B-cell origin. The non-Hodgkin's lymphomasoriginate from components of the spleen, thymus, and lymph nodes. In theWorking Formulation classification scheme, these lymphomas been dividedinto low-, intermediate-, and high-grade categories by virtue of theirnatural histories (see “The Non-Hodgkin's Lymphoma PathologicClassification Project,” Cancer 49(1982):2112-2135). The low-grade orfavorable lymphomas are indolent, with a median survival of 5 to 10years (Horning and Rosenberg (1984) N. Engl. J. Med. 311:1471-1475).Although chemotherapy can induce remissions in the majority of indolentlymphomas, cures are rare, and most patients eventually relapse,requiring further therapy. The intermediate- and high-grade lymphomasare more aggressive tumors, but they have a greater chance for cure withchemotherapy. However, significant numbers of these patients will stillrelapse and require further treatment to induce remissions. Furthermore,patients undergoing chemotherapy can experience toxicity effects.Therefore, there is a need for new therapies for treating diseases ofmalignant B cells.

SUMMARY OF THE INVENTION

Methods for treating a patient with a disease comprising malignant Bcells, including lymphomas such as, non-Hodgkin's lymphomas (high-gradelymphomas, intermediate-grade lymphomas, and low-grade lymphomas),Hodgkin's disease, acute lymphoblastic leukemias, myelomas, chroniclymphocytic leukemias, and myeloblastic leukemias are provided. Themethod comprises treating the patient with anti-CD40 antibodies orantigen-binding fragments thereof that are free of significant agonistactivity when bound to a CD40 antigen on a normal human B cells and thatexhibit antagonist activity when bound to a CD40 antigen on a malignanthuman B cell. Monoclonal antibodies and antigen-binding fragmentsthereof that are suitable for use in the methods of the inventionexhibit the following characteristics: 1) are capable of specificallybinding to a human CD40 antigen expressed on the surface of a humancell; 2) are free of significant agonist activity when bound to a CD40antigen on a normal human B cell; and, 3) exhibit antagonist activitywhen bound to a CD40 antigen on a malignant human B cell. In someembodiments, the anti-CD40 antibody or fragment thereof also exhibitsantagonist activity when bound to CD40 antigen on normal human B cells.The monoclonal antibodies have a strong affinity for CD40 and arecharacterized by a dissociation constant (K_(d)) of at least 10⁻⁵ M,preferably at least about 10⁻⁸ M to about 10⁻²⁰ M, more preferably atleast about 5×10⁻⁹ to about 10⁻¹⁶ M. Suitable monoclonal antibodies havehuman constant regions; preferably they also have wholly or partiallyhumanized framework regions; and most preferably are fully humanantibodies or antigen-binding fragments thereof. Examples of suchmonoclonal antibodies are the antibody designated herein as 15B8, themonoclonal antibody produced by the hybridoma cell line designated 15B8,a monoclonal antibody comprising an amino acid sequence selected fromthe group consisting of SEQ ID NO:2 and SEQ ID NO:4; a monoclonalantibody comprising an amino acid sequence encoded by a nucleic acidmolecule comprising a nucleotide sequence selected from the groupconsisting of SEQ ID NO:1 and SEQ ID NO:3; and antigen-binding fragmentsof these monoclonal antibodies that retain the capability ofspecifically binding to human CD40.

In one embodiment of the invention, the therapy comprises administeringto a patient a therapeutically effective dose of a pharmaceuticalcomposition comprising suitable anti-CD40 antibodies or antigen-bindingfragments thereof. A therapeutically effective dose of the anti-CD40antibody or fragment thereof is in the range from about 0.01 mg/kg toabout 40 mg/kg, from about 0.01 mg/kg to about 30 mg/kg, from about 0.1mg/kg to about 30 mg/kg, from about 1 mg/kg to about 30 mg/kg, fromabout 3 mg/kg to about 30 mg/kg, from about 3 mg/kg to about 25 mg/kg,from about 3 mg/kg to about 20 mg/kg, from about 5 mg/kg to about 15mg/kg, or from about 7 mg/kg to about 12 mg/kg. It is recognized thatthe treatment may comprise administration of a single therapeuticallyeffective dose or administration of multiple therapeutically effectivedoses of the anti-CD40 antibody or antigen-binding fragment thereof.

The anti-CD40 antibodies suitable for use in the methods of theinvention may be modified. Modifications of the anti-CD40 antibodiesinclude, but are not limited to, immunologically active chimericanti-CD40 antibodies, humanized anti-CD40 antibodies, andimmunologically active murine anti-CD40 antibodies.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts representative results of the effect of agonist (MS81)and antagonist (15B8) anti-CD40 antibodies at a concentration of 1, 2,or 5 μg/ml on the proliferation of non-Hodgkin's lymphoma (NHL) cells invitro in the absence of interleukin-4 (IL-4). Malignant B cells wereobtained from tumor infiltrated lymph nodes of a NHL patient. FACSanalysis of the NHL cells confirmed that these cells expressed CD40 andbound the antagonist anti-CD40 antibody 15B8. See Example 3 below fordetails.

FIG. 2 depicts representative results of the effect of agonist (MS81)and antagonist (15B8) anti-CD40 antibodies at a concentration of 1, 2,or 5 μg/ml on the proliferation of non-Hodgkin's lymphoma (NHL) cells invitro in the presence of IL-4 (2 ng/ml). Malignant B cells were obtainedfrom tumor infiltrated lymph nodes of a NHL patient. FACS analysis ofthe NHL cells confirmed that these cells expressed CD40 and bound theantagonist anti-CD40 antibody. See Example 3 below for details.

FIG. 3 depicts representative results of the effect of agonist (MS81)and antagonist (15B8) anti-CD40 antibodies at a concentration of 1, 2,or 5 μg/ml on CD40L-stimulated proliferation of NHL cells in vitro inthe absence of IL-4. The NHL cells were obtained from aRituximab-sensitive NHL patient. See Example 4 below for details.

FIG. 4 depicts a representative dose response curve for the antagonistanti-CD40antibody 15B8 on proliferation of NHL cells stimulated in vitroby CD40L and IL-4(2 ng/ml). The NHL cells were obtained from aRituximab-sensitive NHL patient. See Example 4 below for details.

FIG. 5 depicts dose response curves for the antagonist anti-CD40antibody 15B8 on proliferation of purified human peripheral blood Bcells stimulated in vitro in a CD40L-expressing CHO cell-mediated humanB-cell proliferation assay. The B cells were obtained from 3 healthyindividuals. See Example 6 below for details.

FIG. 6 depicts the effect on the peripheral B-cell count in malechimpanzees after administration of 15B8 at doses of 0.03 or 3 mg/kg.Each dosage level was intravenously administered to 3 chimpanzees, andthe average peripheral B-cell count (per μl) was determined (righty-axis). The mean concentration of 15B8 in the serum (ng/ml) is depictedon the left y-axis. Time measured in days relative to the IVadministration is shown on the x-axis. After administration of 15B8 at 3mg/kg, serum 15B8 concentrations declined in a triphasic pattern with ashort distribution phase, a log-linear elimination phase, and anon-linear elimination phase. The half-life during the log-linearelimination phase was approximately 4 days. Peripheral B-cell numbersdecreased immediately after 15B8 administration and recovered within 3-4weeks. See Example 9 below for details.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed to methods for treating human patientswith diseases that originate from malignant B cells. The methods involvetreatment with an anti-CD40 antibody or antigen-binding fragmentthereof, where administration of the antibody or antigen-bindingfragment thereof promotes a positive therapeutic response within thepatient undergoing this method of therapy. Anti-CD40 antibodies suitablefor use in the methods of the invention have the followingcharacteristics: 1) they specifically bind a human CD40 antigenexpressed on the surface of a human cell; 2) they are free ofsignificant agonist activity when bound to a CD40 antigen on a normalhuman B cell; and 3) they exhibit antagonist activity when bound to aCD40 antigen on a malignant human B cell. These anti-CD40 antibodies andantigen-binding fragments thereof are referred to herein as antagonistanti-CD40 antibodies. Such antibodies include, but are not limited to,the fully human monoclonal antibody 15B8 described below and monoclonalantibodies having the binding characteristics of monoclonal antibody15B8. As discussed in more detail below, these antibodies are specificto CD40 receptors. When these antibodies bind CD40 displayed on thesurface of normal human B cells, the antibodies are free of significantagonist activity; in some embodiments, their binding to CD40 displayedon the surface of normal human B cells results in inhibition ofproliferation and differentiation of these normal human B cells. Thus,the antagonist anti-CD40 antibodies suitable for use in the methods ofthe invention include those monoclonal antibodies that can exhibitantagonist activity toward normal human B cells expressing thecell-surface CD40 antigen. When antagonist anti-CD40 antibodies bindCD40 displayed on the surface of malignant human B cells, the antibodiesexhibit antagonist activity as defined elsewhere herein.

“Treatment” is herein defined as the application or administration of anantagonist anti-CD40 antibody or antigen-binding fragment thereof to apatient, or application or administration of an antagonist anti-CD40antibody or fragment thereof to an isolated tissue or cell line from apatient, where the patient has a disease, a symptom of a disease, or apredisposition toward a disease, where the purpose is to cure, heal,alleviate, relieve, alter, remedy, ameliorate, improve, or affect thedisease, the symptoms of the disease, or the predisposition toward thedisease. By “treatment” is also intended the application oradministration of a pharmaceutical composition comprising the antagonistanti-CD40 antibodies or fragments thereof to a patient, or applicationor administration of a pharmaceutical composition comprising theanti-CD40 antibodies or fragments thereof to an isolated tissue or cellline from a patient, who has a disease, a symptom of a disease, or apredisposition toward a disease, where the purpose is to cure, heal,alleviate, relieve, alter, remedy, ameliorate, improve, or affect thedisease, the symptoms of the disease, or the predisposition toward thedisease.

By “anti-tumor activity” is intended a reduction in the rate ofmalignant B-cell proliferation or accumulation, and hence a decline ingrowth rate of an existing tumor or in a tumor that arises duringtherapy, and/or destruction of existing neoplastic (tumor) cells ornewly formed neoplastic cells, and hence a decrease in the overall sizeof a tumor during therapy. Therapy with at least one anti-CD40 antibody(or antigen-binding fragment thereof) causes a physiological responsethat is beneficial with respect to treatment of disease statescomprising malignant B cells in a human.

The monoclonal antibody 15B8 represents a suitable antagonist anti-CD40antibody for use in the methods of the present invention. This antibodyis described in U.S. Provisional Application Ser. No. 60/237,556, titled“Human Anti-CD40 Antibodies,” filed Oct. 2, 2000, and PCT InternationalApplication No. PCT/US01/30857, also titled “Human Anti-CD40Antibodies,” filed Oct. 2, 2001 (Attorney Docket No. PP 16092.003), bothof which are herein incorporated by reference in their entirety. The15B8 antibody is a fully human anti-CD40 monoclonal antibody of the IgG₂isotype produced from the hybridoma cell line 15B8. The cell line wascreated using splenocytes from an immunized xenotypic mouse containing ahuman immunoglobulin locus (Abgenix). The spleen cells were fused withthe mouse myeloma SP2/0 cells (Sierra BioSource). The resultinghybridomas were sub-cloned several times to create the stable monoclonalcell line 15B8. The hybridoma cell line 15B8 was deposited with theAmerican Type Culture Collection (ATCC), 10801 University Boulevard,Manassas, Va., USA, on Oct. 25, 2001, under the terms of the BudapestTreaty and assigned Patent Deposit_(—) Designation PTA-3814.

The 15B8 cell line was adapted to grow in protein-free medium and usedto create a Master Cell Bank. The Master Cell Bank was tested foridentity and adventitious and endogenous contaminants. The Master CellBank was used to manufacture the desired human IgG₂. The respective 15B8antibody was purified using chromatography and filtration procedures.

The anti-CD40 antibody 15B8 is a polypeptide composed of 1,284 aminoacid residues with a predicted molecular weight of 149,755 with twoheavy chains and two light chains in a heterodimeric arrangement. Aminoacid analysis reveals that the antibody is composed of equimolar amountsof heavy and light chains. The nucleotide and amino acid sequences forthe variable region for the light chain are set forth in SEQ ID NO:1 andSEQ ID NO:2, respectively. The nucleotide and amino acid sequences forthe variable region for the heavy chain are set forth in SEQ ID NO:3 andSEQ ID NO:4, respectively. The 15B8 monoclonal antibody binds solubleCD40 in ELISA-type assays. When tested in vitro for effects onproliferation of B cells from numerous primates, 15B8 acts as anagonistic anti-CD40 antibody in cynomologus, baboon, and rhesus monkeys.In assays with humans, chimpanzees, and marmosets, 15B8 is an antagonistanti-CD40 antibody. The binding affinity of 15B8 to human CD40 is3.1×10⁻⁹M as determined by the Biacore™ assay.

Suitable antagonist anti-CD40 antibodies for use in the methods of thepresent invention exhibit a strong single-site binding affinity for theCD40 cell-surface antigen. The monoclonal antibodies of the inventionexhibit a dissociation constant (K_(d)) for CD40 of at least 10⁻⁵ M, atleast 3×10⁻⁵ M, preferably at least 10⁻⁶ M to 10⁻⁷ M, more preferably atleast 10⁻⁸ M to about 10⁻²⁰ M, yet more preferably at least 5×10⁻⁹ M toabout 10⁻¹⁸ M, most preferably at least about 5×10⁻⁹ M to about 10⁻¹⁶ M,such as 10⁻⁸ M, 5×10⁻⁹ M, 10⁻⁹ M, 5×10⁻¹⁰ M, 10⁻¹⁰ M, 5×10⁻¹¹ M, 10⁻¹¹M, 5×10⁻¹² M, 10⁻¹² M, 5×10⁻¹³ M, 10⁻¹³ M, 5×10⁻¹⁴ M, 10⁻¹⁴ M, 5×10⁻¹⁵M, 10⁻¹⁵ M, 5×10⁻¹⁶ M, or 10⁻¹⁶ M, as measured using a standard assaysuch as Biacore™.

Biacore™ analysis is known in the art and details are provided in the“BIAapplications handbook.”

By “CD40 antigen” is intended a glycosylated transmembrane peptide orany fragment thereof (GenBank Accession No. X60592; U.S. Pat. Nos.5,674,492 and 4,708,871; Stamenkovic et al. (1989) EMBO 8:1403; Clark(1990) Tissue Antigens 36:33; Barclay et al. (1997) The LeucocyteAntigen Facts Book (2d ed.; Academic Press, San Diego)). The CD40receptor is displayed on the surface of a variety of cell types, asdescribed elsewhere herein. By “displayed on the surface” and “expressedon the surface” is intended that all or a portion of the CD40 antigen isexposed to the exterior of the cell. The displayed or expressed CD40antigen may be fully or partially glycosylated.

By “agonist activity” is intended that the substance functions as anagonist. An agonist combines with a receptor on a cell and initiates areaction or activity that is similar to or the same as that initiated bythe receptor's natural ligand. An agonist of CD40 induces any or all of,but not limited to, the following responses: B-cell proliferation anddifferentiation, antibody production, intercellular adhesion, B-cellmemory generation, isotype switching, up-regulation of cell-surfaceexpression of MHC Class II and CD80/86, and secretion ofpro-inflammatory cytokines such as IL-8, IL-12, and TNF. By “antagonistactivity” is intended that the substance functions as an antagonist. Anantagonist of CD40 prevents or reduces induction of any of the responsesinduced by binding of the CD40 receptor to an agonist ligand,particularly CD40L. The antagonist may reduce induction of any one ormore of the responses to agonist binding by 5%, 10%, 15%, 20%, 25%, 30%,35%, preferably 40%, 45%, 50%, 55%, 60%, more preferably 70%, 80%, 85%,and most preferably 90%, 95%, 99%, or 100%. Methods for measuring B-cellresponses are known to one of skill in the art and include, but are notlimited to, B-cell proliferation assays, Banchereau-Like-B-Cellproliferation assays, T-cell helper assays for antibody production,co-stimulation of B-cell proliferation assays, and assays forup-regulation of B-cell activation markers. Several of these assays arediscussed in more detail elsewhere herein.

By “significant” agonist activity is intended an agonist activity of atleast 30%, 35%, 40%, 45%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or100% greater than the agonist activity induced by a neutral substance ornegative control as measured in an assay of a B-cell response. Asubstance “free of significant agonist activity” would exhibit anagonist activity of not more than about 25% greater than the agonistactivity induced by a neutral substance or negative control, preferablynot more than about 20% greater, 15% greater, 10% greater, 5% greater,1% greater, 0.5% greater, or even not more than about 0.1% greater thanthe agonist activity induced by a neutral substance or negative controlas measured in an assay of a B-cell response. The antagonist anti-CD40antibodies useful in the methods of the present invention are free ofsignificant agonist activity as noted above when bound to a CD40 antigenon a normal human B cell. In one embodiment of the invention, theantagonist anti-CD40 antibody is free of significant agonist activity inone B-cell response. In another embodiment of the invention, theantagonist anti-CD40 antibody is free of significant agonist activity inassays of more than one B-cell response (e.g., proliferation anddifferentiation, or proliferation, differentiation, and antibodyproduction).

As used herein “anti-CD40 antibody” encompasses any antibody thatspecifically recognizes the CD40 B-cell surface antigen, includingpolyclonal antibodies, monoclonal antibodies, single-chain antibodies,and fragments thereof such as Fab, F(ab′)₂, F_(v), and other fragmentswhich retain the antigen binding function of the parent anti-CD40antibody. Polyclonal sera may be prepared by conventional methods. Ingeneral, a solution containing the CD40 antigen is first used toimmunize a suitable animal, preferably a mouse, rat, rabbit, or goat.Rabbits or goats are preferred for the preparation of polyclonal seradue to the volume of serum obtainable, and the availability of labeledanti-rabbit and anti-goat antibodies. Polyclonal sera can be prepared ina transgenic animal, preferably a mouse bearing human immunoglobulinloci. In a preferred embodiment, Sf9 cells expressing CD40 are used asthe immunogen. Immunization can also be performed by mixing oremulsifying the antigen-containing solution in saline, preferably in anadjuvant such as Freund's complete adjuvant, and injecting the mixtureor emulsion parenterally (generally subcutaneously or intramuscularly).A dose of 50-200 μg/injection is typically sufficient. Immunization isgenerally boosted 2-6 weeks later with one or more injections of theprotein in saline, preferably using Freund's incomplete adjuvant. Onemay alternatively generate antibodies by in vitro immunization usingmethods known in the art, which for the purposes of this invention isconsidered equivalent to in vivo immunization. Polyclonal antisera areobtained by bleeding the immunized animal into a glass or plasticcontainer, incubating the blood at 25° C. for one hour, followed byincubating at 4° C. for 2-18 hours. The serum is recovered bycentrifugation (e.g., 1,000×g for 10 minutes). About 20-50 ml per bleedmay be obtained from rabbits.

Preferably the antibody is monoclonal in nature. By “monoclonalantibody” is intended an antibody obtained from a population ofsubstantially homogeneous antibodies, i.e., the individual antibodiescomprising the population are identical except for possible naturallyoccurring mutations that may be present in minor amounts. Monoclonalantibodies are highly specific, being directed against a singleantigenic site, i.e., the CD40 B-cell surface antigen in the presentinvention. Furthermore, in contrast to conventional (polyclonal)antibody preparations that typically include different antibodiesdirected against different determinants (epitopes), each monoclonalantibody is directed against a single determinant on the antigen. Themodifier “monoclonal” indicates the character of the antibody as beingobtained from a substantially homogeneous population of antibodies, andis not to be construed as requiring production of the antibody by anyparticular method. For example, the monoclonal antibodies to be used inaccordance with the present invention may be made by the hybridomamethod first described by Kohler et al. (1975) Nature 256:495, or may bemade by recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567).The “monoclonal antibodies” may also be isolated from phage antibodylibraries using the techniques described in, for example, Clackson etal. (1991) Nature 352:624-628; Marks et al. (1991) J. Mol. Biol.222:581-597; and U.S. Pat. No. 5,514,548.

Monoclonal antibodies can be prepared using the method of Kohler et al.(1975) Nature 256:495-496, or a modification thereof. Typically, a mouseis immunized with a solution containing an antigen. Immunization can beperformed by mixing or emulsifying the antigen-containing solution insaline, preferably in an adjuvant such as Freund's complete adjuvant,and injecting the mixture or emulsion parenterally. Any method ofimmunization known in the art may be used to obtain the monoclonalantibodies of the invention. After immunization of the animal, thespleen (and optionally, several large lymph nodes) are removed anddissociated into single cells. The spleen cells may be screened byapplying a cell suspension to a plate or well coated with the antigen ofinterest. The B cells expressing membrane bound immunoglobulin specificfor the antigen bind to the plate and are not rinsed away. Resulting Bcells, or all dissociated spleen cells, are then induced to fuse withmyeloma cells to form hybridomas, and are cultured in a selectivemedium. The resulting cells are plated by serial dilution and areassayed for the production of antibodies that specifically bind theantigen of interest (and that do not bind to unrelated antigens). Theselected monoclonal antibody (mAb)-secreting hybridomas are thencultured either in vitro (e.g., in tissue culture bottles or hollowfiber reactors), or in vivo (as ascites in mice).

As an alternative to the use of hybridomas, antibody can be produced ina cell line such as a CHO cell line, as disclosed in U.S. Pat. Nos.5,545,403; 5,545,405; and 5,998,144; incorporated herein by reference.Briefly the cell line is transfected with vectors capable of expressinga light chain and a heavy chain, respectively. By transfecting the twoproteins on separate vectors, chimeric antibodies can be produced.Another advantage is the correct glycosylation of the antibody.

Monoclonal antibodies to CD40 are known in the art. See, for example,the sections dedicated to B-cell antigen in McMichael, ed. (1987; 1989)Leukocyte Typing III and IV (Oxford University Press, New York); U.S.Pat. Nos. 5,674,492; 5,874,082; 5,677,165; 6,056,959; WO 00/63395;copending U.S. Provisional Patent Application Ser. No. 60/237,556,titled, “Human Anti-CD40 Antibodies,” filed Oct. 2, 2000; Gordon et al.(1988) J. Immunol. 140:1425; Valle et al. (1989) Eur. J. Immunol.19:1463; Clark et al. (1986) PNAS 83:4494; Paulie et al. (1989) J.Immunol. 142:590; Gordon et al. (1987) Eur. J. Immunol. 17:1535; Jabaraet al. (1990) J. Exp. Med. 172:1861; Zhang et al. (1991) J. Immunol.146:1836; Gascan et al. (1991) J. Immunol. 147:8; Banchereau et al.(1991) Clin. Immunol. Spectrum 3:8; and Banchereau et al. (1991) Science251:70; all of which are herein incorporated by reference.

Additionally, the term “anti-CD40 antibody” as used herein encompasseschimeric anti-CD40 antibodies. By “chimeric” antibodies is intendedantibodies that are most preferably derived using recombinantdeoxyribonucleic acid techniques and which comprise both human(including immunologically “related” species, e.g., chimpanzee) andnon-human components. Thus, the constant region of the chimeric antibodyis most preferably substantially identical to the constant region of anatural human antibody; the variable region of the chimeric antibody ismost preferably derived from a non-human source and has the desiredantigenic specificity to the CD40 cell-surface antigen. The non-humansource can be any vertebrate source that can be used to generateantibodies to a human CD40 cell-surface antigen or material comprising ahuman CD40 cell-surface antigen. Such non-human sources include, but arenot limited to, rodents (e.g., rabbit, rat, mouse, etc.; see, forexample, U.S. Pat. No. 4,816,567, herein incorporated by reference) andnon-human primates (e.g., Old World Monkey, Ape, etc.; see, for example,U.S. Pat. Nos. 5,750,105 and 5,756,096; herein incorporated byreference). As used herein, the phrase “immunologically active” whenused in reference to chimeric anti-CD40 antibodies means a chimericantibody that binds human CD40.

Humanized anti-CD40 antibodies are also encompassed by the termanti-CD40 antibody as used herein. By “humanized” is intended forms ofanti-CD40 antibodies that contain minimal sequence derived fromnon-human immunoglobulin sequences. For the most part, humanizedantibodies are human immunoglobulins (recipient antibody) in whichresidues from a hypervariable region (also known as complementaritydetermining region or CDR) of the recipient are replaced by residuesfrom a hypervariable region of a non-human species (donor antibody) suchas mouse, rat, rabbit, or nonhuman primate having the desiredspecificity, affinity, and capacity. Humanization can be essentiallyperformed following the method of Winter and co-workers (Jones et al.(1986) Nature 321:522-525; Riechmann et al. (1988) Nature 332:323-327;Verhoeyen et al. (1988) Science 239:1534-1536), by substituting rodentor mutant rodent CDRs or CDR sequences for the corresponding sequencesof a human antibody. See also U.S. Pat. Nos. 5,225,539; 5,585,089;5,693,761; 5,693,762; 5,859,205; herein incorporated by reference. Insome instances, residues within the framework regions of one or morevariable regions of the human immunoglobulin are replaced bycorresponding non-human residues (see, for example, U.S. Pat. Nos.5,585,089; 5,693,761; 5,693,762; and 6,180,370). Furthermore, humanizedantibodies may comprise residues that are not found in the recipientantibody or in the donor antibody. These modifications are made tofurther refine antibody performance (e.g., to obtain desired affinity).In general, the humanized antibody will comprise substantially all of atleast one, and typically two, variable domains, in which all orsubstantially all of the hypervariable regions correspond to those of anon-human immunoglobulin and all or substantially all of the frameworkregions are those of a human immunoglobulin sequence. The humanizedantibody optionally also will comprise at least a portion of animmunoglobulin constant region (Fc), typically that of a humanimmunoglobulin. For further details see Jones et al. (1986) Nature331:522-525; Riechmann et al. (1988) Nature 332:323-329; and Presta(1992) Curr. Op. Struct. Biol. 2:593-596; herein incorporated byreference. Accordingly, such “humanized” antibodies may includeantibodies wherein substantially less than an intact human variabledomain has been substituted by the corresponding sequence from anon-human species. In practice, humanized antibodies are typically humanantibodies in which some CDR residues and possibly some frameworkresidues are substituted by residues from analogous sites in rodentantibodies. See, for example, U.S. Pat. Nos. 5,225,539; 5,585,089;5,693,761; 5,693,762; 5,859,205. See also U.S. Pat. No. 6,180,370, andInternational Publication No. WO 01/27160, where humanized antibodiesand techniques for producing humanized antibodies having improvedaffinity for a predetermined antigen are disclosed.

Also encompassed by the term anti-CD40 antibodies are xenogeneic ormodified anti-CD40 antibodies produced in a non-human mammalian host,more particularly a transgenic mouse, characterized by inactivatedendogenous immunoglobulin (Ig) loci. In such transgenic animals,competent endogenous genes for the expression of light and heavysubunits of host immunoglobulins are rendered non-functional andsubstituted with the analogous human immunoglobulin loci. Thesetransgenic animals produce human antibodies in the substantial absenceof light or heavy host immunoglobulin subunits. See, for example, U.S.Pat. Nos. 5,877,397 and 5,939,598, herein incorporated by reference.

Fragments of the anti-CD40 antibodies are suitable for use in themethods of the invention so long as they retain the desired affinity ofthe full-length antibody. Thus, a fragment of an anti-CD40 antibody willretain the ability to bind to the CD40 B-cell surface antigen. Suchfragments are characterized by properties similar to the correspondingfull-length antagonist anti-CD40 antibody, that is the fragments will 1)specifically bind a human CD40 antigen expressed on the surface of ahuman cell; 2) are free of significant agonist activity when bound to aCD40 antigen on a normal human B cell; and 3) exhibit antagonistactivity when bound to a CD40 antigen on a malignant human B cell. Wherethe full-length antagonist anti-CD40 antibody exhibits antagonistactivity when bound to the CD40 antigen on the surface of a normal humanB cell, the fragment will also exhibit such antagonist activity. Suchfragments are referred to herein as “antigen-binding” fragments.

Suitable antigen-binding fragments of an antibody comprise a portion ofa full-length antibody, generally the antigen-binding or variable regionthereof. Examples of antibody fragments include, but are not limited to,Fab, F(ab′)₂, and Fv fragments and single-chain antibody molecules. By“single-chain Fv” or “sFv” antibody fragments is intended fragmentscomprising the V_(H) and V_(L) domains of an antibody, wherein thesedomains are present in a single polypeptide chain. See, for example,U.S. Pat. Nos. 4,946,778; 5,260,203; 5,455,030; 5,856,456; hereinincorporated by reference. Generally, the Fv polypeptide furthercomprises a polypeptide linker between the V_(H) and V_(L) domains thatenables the sFv to form the desired structure for antigen binding. For areview of sFv see Pluckthun (1994) in The Pharmacology of MonoclonalAntibodies, Vol. 113, ed. Rosenburg and Moore (Springer-Verlag, NewYork), pp. 269-315.

Antibodies or antibody fragments can be isolated from antibody phagelibraries generated using the techniques described in, for example,McCafferty et al. (1990) Nature 348:552-554 (1990) and U.S. Pat. No.5,514,548. Clackson et al. (1991) Nature 352:624-628 and Marks et al.(1991) J. Mol. Biol. 222:581-597 describe the isolation of murine andhuman antibodies, respectively, using phage libraries. Subsequentpublications describe the production of high affinity (nM range) humanantibodies by chain shuffling (Marks et al. (1992) Bio/Technology10:779-783), as well as combinatorial infection and in vivorecombination as a strategy for constructing very large phage libraries(Waterhouse et al. (1993) Nucleic. Acids Res. 21:2265-2266). Thus, thesetechniques are viable alternatives to traditional monoclonal antibodyhybridoma techniques for isolation of monoclonal antibodies.

Various techniques have been developed for the production of antibodyfragments. Traditionally, these fragments were derived via proteolyticdigestion of intact antibodies (see, e.g., Morimoto et al. (1992)Journal of Biochemical and Biophysical Methods 24:107-117 (1992) andBrennan et al. (1985) Science 229:81). However, these fragments can nowbe produced directly by recombinant host cells. For example, theantibody fragments can be isolated from the antibody phage librariesdiscussed above. Alternatively, Fab′-SH fragments can be directlyrecovered from E. coli and chemically coupled to form F(ab′)₂ fragments(Carter et al. (1992) Bio/Technology 10:163-167). According to anotherapproach, F(ab′)₂ fragments can be isolated directly from recombinanthost cell culture. Other techniques for the production of antibodyfragments will be apparent to the skilled practitioner.

Antagonist anti-CD40 antibodies useful in the methods of the presentinvention include the 15B8 monoclonal antibody disclosed herein as wellas antibodies differing from this antibody but retaining the CDRs; andantibodies with one or more amino acid addition(s), deletion(s), orsubstitution(s), wherein the antagonist activity is measured byinhibition of malignant B cell proliferation and/or differentiation. Theinvention also encompasses de-immunized antagonist anti-CD40 antibodies,which can be produced as described in, for example, InternationalPublication Nos. WO 98/52976 and WO 0034317; herein incorporated byreference. In this manner, residues within the antagonist anti-CD40antibodies of the invention are modified so as to render the antibodiesnon- or less immunogenic to humans while retaining their antagonistactivity toward malignant human B cells, wherein such activity ismeasured by assays noted elsewhere herein. Also included within thescope of the claims are fusion proteins comprising an antagonistanti-CD40 antibody of the invention, or a fragment thereof, which fusionproteins can be synthesized or expressed from correspondingpolynucleotide vectors, as is known in the art. Such fusion proteins aredescribed with reference to conjugation of antibodies as noted below.

The antibodies of the present invention can have sequence variationsproduced using methods described in, for example, Patent PublicationNos. EP 0 983 303 A1, WO 00/34317, and WO 98/52976, incorporated hereinby reference. For example, it has been shown that sequences within theCDR can cause an antibody to bind to MHC Class II and trigger anunwanted helper T cell response. A conservative substitution can allowthe antibody to retain binding activity yet lose its ability to triggeran unwanted T cell response. Any such conservative or non-conservativesubstitutions can be made using art-recognized methods, such as thosenoted elsewhere herein, and the resulting antibodies will fall withinthe scope of the invention. The variant antibodies can be routinelytested for antagonist activity, affinity, and specificity using methodsdescribed herein.

An antibody produced by any of the methods described above, or any othermethod not disclosed herein, will fall within the scope of the inventionif it possesses at least one of the following biological activities:inhibition of immunoglobulin secretion by normal human peripheral Bcells stimulated by T cells; inhibition of proliferation of normal humanperipheral B cells stimulated by Jurkat T cells; inhibition ofproliferation of normal human peripheral B cells stimulated byCD40L-expressing cells; and inhibition of proliferation of humanmalignant B cells as noted below. These assays can be performed asdescribed in the Examples herein. See also the assays described inSchultze et al. (1998) Proc. Natl. Acad. Sci. USA 92:8200-8204; Dentonet al. (1998) Pediatr. Transplant. 2:6-15; Evans et al. (2000) J.Immunol. 164:688-697; Noelle (1998) Agents Actions Suppl. 49:17-22;Lederman et al. (1996) Curr. Opin. Hematol. 3:77-86; Coligan et al.(1991) Current Protocols in Immunology 13:12; Kwekkeboom et al. (1993)Immunology 79:439-444; and U.S. Pat. Nos. 5,674,492 and 5,847,082;herein incorporated by reference.

Any of the previously described antagonist anti-CD40 antibodies orantibody fragments thereof may be conjugated prior to use in the methodsof the present invention. Methods for producing conjugated antibodiesare known in the art. Thus, the anti-CD40 antibody may be labeled usingan indirect labeling or indirect labeling approach. By “indirectlabeling” or “indirect labeling approach” is intended that a chelatingagent is covalently attached to an antibody and at least oneradionuclide is inserted into the chelating agent. See, for example, thechelating agents and radionuclides described in Srivagtava and Mease(1991) Nucl. Med. Bio. 18:589-603, herein incorporated by reference.Alternatively, the anti-CD40 antibody may be labeled using “directlabeling” or a “direct labeling approach”, where a radionuclide iscovalently attached directly to an antibody (typically via an amino acidresidue). Preferred radionuclides are provided in Srivagtava and Mease(1991) supra. The indirect labeling approach is particularly preferred.See also, for example, International Publication Nos. WO 00/52031 and WO00/52473, where a linker is used to attach a radioactive label toantibodies; and the labeled forms of anti-CD40 antibodies described inU.S. Pat. No. 6,015,542; herein incorporated by reference.

Further, an antibody (or fragment thereof) may be conjugated to atherapeutic moiety such as a cytotoxin, a therapeutic agent, or aradioactive metal ion. A cytotoxin or cytotoxic agent includes any agentthat is detrimental to cells. Examples include taxol, cytochalasin B,gramicidin D, ethidium bromide, emetine, mitomycin, etoposide,tenoposide, vincristine, vinblastine, colchicin, doxorubicin,daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin,actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine,tetracaine, lidocaine, propranolol, and puromycin and analogs orhomologs thereof. Therapeutic agents include, but are not limited to,antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine,cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g.,mechlorethamine, thioepa chlorambucil, melphalan, carmustine (BSNU) andlomustine (CCNU), cyclothosphamide, busulfan, dibromomannitol,streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (II) (DDP)cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) anddoxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin),bleomycin, mithramycin, and anthramycin (AMC)), and anti-mitotic agents(e.g., vincristine and vinblastine). The conjugates of the invention canbe used for modifying a given biological response; the drug moiety isnot to be construed as limited to classical chemical therapeutic agents.For example, the drug moiety may be a protein or polypeptide possessinga desired biological activity. Such proteins may include, for example, atoxin such as abrin, ricin A, pseudomonas exotoxin, or diphtheria toxin;a protein such as tumor necrosis factor, interferon-alpha,interferon-beta, nerve growth factor, platelet derived growth factor,tissue plasminogen activator; or, biological response modifiers such as,for example, lymphokines, interleukin-1 (“IL-1”), interleukin-2(“IL-2”), interleukin-6 (“IL-6”), granulocyte macrophase colonystimulating factor (“GM-CSF”), granulocyte colony stimulating factor(“G-CSF”), or other growth factors.

Techniques for conjugating such therapeutic moiety to antibodies arewell known. See, for example, Arnon et al. (1985) “Monoclonal Antibodiesfor Immunotargeting of Drugs in Cancer Therapy,” in MonoclonalAntibodies and Cancer Therapy, ed. Reisfeld et al. (Alan R. Liss, Inc.),pp. 243-256; ed. Hellstrom et al. (1987) “Antibodies for Drug Delivery,”in Controlled Drug Delivery, ed. Robinson et al. (2d ed; Marcel Dekker,Inc.), pp. 623-653; Thorpe (1985) “Antibody Carriers of Cytotoxic Agentsin Cancer Therapy: A Review,” in Monoclonal Antibodies '84: Biologicaland Clinical Applications, ed. Pinchera et al. pp. 475-506 (EditriceKurds, Milano, Italy, 1985); “Analysis, Results, and Future Prospectiveof the Therapeutic Use of Radiolabeled Antibody in Cancer Therapy,” inMonoclonal Antibodies for Cancer Detection and Therapy, ed. Baldwin etal. (Academic Press, New York, 1985), pp. 303-316; and Thorpe et al.(1982) Immunol. Rev. 62:119-158.

Alternatively, an antibody can be conjugated to a second antibody toform an antibody heteroconjugate as described by Segal in U.S. Pat. No.4,676,980. In addition, linkers may be used between the labels and theantibodies of the invention (see U.S. Pat. No. 4,831,175). Antibodiesor, antigen-binding fragments thereof may be directly labeled withradioactive iodine, indium, yttrium, or other radioactive particle knownin the art (U.S. Pat. No. 5,595,721). Treatment may consist of acombination of treatment with conjugated and nonconjugated antibodiesadministered simultaneously or subsequently (WO 00/52031 and WO00/52473).

Methods of the invention are directed to the use of antagonist anti-CD40antibodies to treat patients having a disease comprising malignant Bcells. By “malignant” B cell is intended any neoplastic B cell,including but not limited to B cells derived from lymphomas includinglow-, intermediate-, and high-grade B-cell lymphomas, immunoblasticlymphomas, non-Hodgkin's lymphomas, Hodgkin's disease, Epstein-BarrVirus (EBV) induced lymphomas, and AIDS-related lymphomas, as well asB-cell acute lymphoblastic leukemias, myelomas, chronic lymphocyticleukemias, acute myeloblastic leukemias, and the like.

The methods of the invention find use in the treatment of non-Hodgkin'slymphomas related to abnormal, uncontrollable B cell proliferation oraccumulation. For purposes of the present invention, such lymphomas willbe referred to according to the Working Formulation classificationscheme, that is those B-cell lymphomas categorized as low grade,intermediate grade, and high grade (see “The Non-Hodgkin's LymphomaPathologic Classification Project,” Cancer 49(1982):2112-2135). Thus,low-grade B-cell lymphomas include small lymphocytic, follicularsmall-cleaved cell, and follicular mixed small-cleaved and large celllymphomas; intermediate-grade lymphomas include follicular large cell,diffuse small cleaved cell, diffuse mixed small and large cell, anddiffuse large cell lymphomas; and high-grade lymphomas include largecell immunoblastic, lymphoblastic, and small non-cleaved cell lymphomasof the Burkitt's and non-Burkitt's type.

It is recognized that the methods of the invention are useful in thetherapeutic treatment of B-cell lymphomas that are classified accordingto the Revised European and American Lymphoma Classification (REAL)system. Such B-cell lymphomas include, but are not limited to, lymphomasclassified as precursor B-cell neoplasms, such as B-lymphoblasticleukemia/lymphoma; peripheral B-cell neoplasms, including B-cell chroniclymphocytic leukemia/small lymphocytic lymphoma, lymphoplasmacytoidlymphoma/immunocytoma, mantle cell lymphoma (MCL), follicle centerlymphoma (follicular) (including diffuse small cell, diffuse mixed smalland large cell, and diffuse large cell lymphomas), marginal zone B-celllymphoma (including extranodal, nodal, and splenic types), hairy cellleukemia, plasmacytoma/myeloma, diffuse large cell B-cell lymphoma ofthe subtype primary mediastinal (thymic), Burkitt's lymphoma, andBurkitt's like high grade B-cell lymphoma; acute leukemias; acutelymphocytic leukemias; myeloblastic leukemias; acute myelocyticleukemias; promyelocytic leukemia; myelomonocytic leukemia; monocyticleukemia; erythroleukemia; granulocytic leukemia (chronic myelocyticleukemia); chronic lymphocytic leukemia; polycythemia vera; multiplemyeloma; Waldenstrom's macroglobulinemia; heavy chain disease; andunclassifiable low-grade or high-grade B-cell lymphomas.

It is recognized that the methods of the invention may be useful inpreventing further tumor outgrowths arising during therapy. The methodsof the invention are particularly useful in the treatment of subjectshaving low-grade B-cell lymphomas, particularly those subjects havingrelapses following standard chemotherapy. Low-grade B-cell lymphomas aremore indolent than the intermediate- and high-grade B-cell lymphomas andare characterized by a relapsing/remitting course. Thus, treatment ofthese lymphomas is improved using the methods of the invention, asrelapse episodes are reduced in number and severity.

The antagonist anti-CD40 antibodies described herein may also find usein the treatment of inflammatory diseases and deficiencies or disordersof the immune system including, but not limited to, systemic lupuserythematosus, psoriasis, scleroderma, CREST syndrome, inflammatorymyositis, Sjogren's syndrome, mixed connective tissue disease,rheumatoid arthritis, multiple sclerosis, inflammatory bowel disease,acute respiratory distress syndrome, pulmonary inflammation, idiopathicpulmonary fibrosis, osteoporosis, delayed type hypersensitivity, asthma,primary biliary cirrhosis, and idiopathic thrombocytopenic purpura.

In accordance with the methods of the present invention, at least oneantagonist anti-CD40 antibody (or antigen-binding fragment thereof) asdefined elsewhere herein is used to promote a positive therapeuticresponse with respect to a malignant human B cell. By “positivetherapeutic response” is intended an improvement in the disease inassociation with the anti-tumor activity of these antibodies orfragments thereof, and/or an improvement in the symptoms associated withthe disease. That is, an anti-proliferative effect, the prevention offurther tumor outgrowths, and/or a decrease in B symptoms can beobserved. Thus, for example, an improvement in the disease may becharacterized as a complete response. By “complete response” is intendedan absence of clinically detectable disease with normalization of anypreviously abnormal radiographic studies, bone marrow, and cerebrospinalfluid (CSF). Such a response must persist for at least one monthfollowing treatment according to the methods of the invention.Alternatively, an improvement in the disease may be categorized as beinga partial response. By “partial response” is intended at least about a50% decrease in all measurable tumor burden (i.e., the number of tumorcells present in the subject) in the absence of new lesions andpersisting for at least one month. Such a response is applicable tomeasurable tumors only. In addition to these positive therapeuticresponses, the subject undergoing therapy with the antagonist anti-CD40antibody or antigen-binding fragment thereof may experience thebeneficial effect of an improvement in the symptoms associated with thedisease. Thus the subject may experience a decrease in the so-called Bsymptoms, i.e., night sweats, fever, weight loss, and/or urticaria.

By “therapeutically effective dose or amount” is intended an amount ofantagonist anti-CD40 antibody or antigen-binding fragment thereof that,when administered brings about a positive therapeutic response withrespect to treatment of a patient with a disease comprising malignant Bcells. Administration of the pharmaceutical composition comprising thetherapeutically effective dose or amount can be achieved using anyacceptable administration method known in the art. Preferably thepharmaceutical composition comprising the antagonist anti-CD40 antibodyor antigen-binding fragment thereof is administered intravenously,preferably by infusion over a period of about 1 to about 10 hours, morepreferably over about 1 to about 8 hours, even more preferably overabout 2 to about 7 hours, still more preferably over about 4 to about 6hours, depending upon the anti-CD40 antibody being administered. Theinitial infusion with the pharmaceutical composition may be given over aperiod of about 4 to about 6 hours with subsequent infusions deliveredmore quickly. Subsequent infusions may be administered over a period ofabout 1 to about 6 hours, preferably about 1 to about 4 hours, morepreferably about 1 to about 3 hours, yet more preferably about 1 toabout 2 hours.

A pharmaceutical composition of the invention is formulated to becompatible with its intended route of administration. Examples of routesof administration include parenteral, e.g., intravenous, intradermal,subcutaneous, oral (e.g., inhalation), transdermal (topical),transmucosal, and rectal administration. Solutions or suspensions usedfor parenteral, intradermal, or subcutaneous application can include thefollowing components: a sterile diluent such as water for injection,saline solution, fixed oils, polyethylene glycols, glycerin, propyleneglycol or other synthetic solvents; antibacterial agents such as benzylalcohol or methyl parabens; antioxidants such as ascorbic acid or sodiumbisulfite; chelating agents such as ethylenediaminetetraacetic acid;buffers such as acetates, citrates or phosphates and agents for theadjustment of tonicity such as sodium chloride or dextrose. pH can beadjusted with acids or bases, such as hydrochloric acid or sodiumhydroxide. The parenteral preparation can be enclosed in ampoules,disposable syringes, or multiple dose vials made of glass or plastic.

The anti-CD40 antibodies are typically provided by standard techniquewithin a pharmaceutically acceptable buffer, for example, sterilesaline, sterile buffered water, propylene glycol, combinations of theforegoing, etc. Methods for preparing parenterally administrable agentsare described in Remington's Pharmaceutical Sciences (18^(th) ed.; MackPublishing Company, Eaton, Pa., 1990), herein incorporated by reference.See also, for example, WO 98/56418, which describes stabilized antibodypharmaceutical formulations suitable for use in the methods of thepresent invention.

The amount of at least one anti-CD40 antibody or fragment thereof to beadministered is readily determined by one of ordinary skill in the artwithout undue experimentation. Factors influencing the mode ofadministration and the respective amount of at least one antagonistanti-CD40 antibody (or fragment thereof) include, but are not limitedto, the particular lymphoma undergoing therapy, the severity of thedisease, the history of the disease, and the age, height, weight,health, and physical condition of the individual undergoing therapy.Similarly, the amount of antagonist anti-CD40 antibody or fragmentthereof to be administered will be dependent upon the mode ofadministration and whether the subject will undergo a single dose ormultiple doses of this anti-tumor agent. Generally, a higher dosage ofanti-CD40 antibody or fragment thereof is preferred with increasingweight of the patient undergoing therapy. The dose of anti-CD40 antibodyor fragment thereof to be administered is in the range from about 0.003mg/kg to about 50 mg/kg, preferably in the range of 0.01 mg/kg to about40 mg/kg. Thus, for example, the dose can be 0.01 mg/kg, 0.03 mg/kg, 0.1mg/kg, 0.3 mg/kg, 0.5 mg/kg, 1 mg/kg, 1.5 mg/kg, 2 mg/kg, 2.5 mg/kg, 3mg/kg, 5 mg/kg, 7 mg/kg, 10 mg/kg, 15 mg/kg, 20 mg/kg, 25 mg/kg, 30mg/kg, 35 mg/kg, 40 mg/kg, 45 mg/kg, or 50 mg/kg.

In another embodiment of the invention, the method comprisesadministration of multiple doses of antagonist anti-CD40 antibody orfragment thereof. The method may comprise administration of 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, or more therapeuticallyeffective doses of a pharmaceutical composition comprising an antagonistanti-CD40 antibody or fragment thereof. The frequency and duration ofadministration of multiple doses of the pharmaceutical compositionscomprising anti-CD40 antibody or fragment thereof can be readilydetermined by one of skill in the art without undue experimentation.Moreover, treatment of a subject with a therapeutically effective amountof an antibody can include a single treatment or, preferably, caninclude a series of treatments. In a preferred example, a subject istreated with antagonist anti-CD40 antibody or antigen-binding fragmentthereof in the range of between about 0.1 to 20 mg/kg body weight, onceper week for between about 1 to 10 weeks, preferably between about 2 to8 weeks, more preferably between about 3 to 7 weeks, and even morepreferably for about 4, 5, or 6 weeks. Treatment may occur annually toprevent relapse or upon indication of relapse. It will also beappreciated that the effective dosage of antibody or antigen-bindingfragment thereof used for treatment may increase or decrease over thecourse of a particular treatment. Changes in dosage may result andbecome apparent from the results of diagnostic assays as describedherein. Thus, in one embodiment, the dosing regimen includes a firstadministration of a therapeutically effective dose of at least oneanti-CD40 antibody or fragment thereof on days 1, 7, 14, and 21 of atreatment period. In another embodiment, the dosing regimen includes afirst administration of a therapeutically effective dose of at least oneanti-CD40 antibody or fragment thereof on days 1, 2, 3, 4, 5, 6, and 7of a week in a treatment period. Further embodiments include a dosingregimen having a first administration of a therapeutically effectivedose of at least one anti-CD40 antibody or fragment thereof on days 1,3, 5, and 7 of a week in a treatment period; a dosing regimen includinga first administration of a therapeutically effective dose of at leastone anti-CD40 antibody or fragment thereof on days 1 and 3 of a week ina treatment period; and a preferred dosing regimen including a firstadministration of a therapeutically effective dose of at least oneanti-CD40 antibody or fragment thereof on day 1 of a week in a treatmentperiod. The treatment period may comprise 1 week, 2 weeks, 3 weeks, amonth, 3 months, 6 months, or a year. Treatment periods may besubsequent or separated from each other by a day, a week, 2 weeks, amonth, 3 months, 6 months, or a year.

The antagonist anti-CD40 antibodies present in the pharmaceuticalcompositions described herein for use in the methods of the inventionmay be native or obtained by recombinant techniques, and may be from anysource, including mammalian sources such as, e.g., mouse, rat, rabbit,primate, pig, and human. Preferably such polypeptides are derived from ahuman source, and more preferably are recombinant, human proteins fromhybridoma cell lines.

The pharmaceutical compositions useful in the methods of the inventionmay comprise biologically active variants of the antagonist anti-CD40antibodies of the invention. Such variants should retain the desiredbiological activity of the native polypeptide such that thepharmaceutical composition comprising the variant polypeptide has thesame therapeutic effect as the pharmaceutical composition comprising thenative polypeptide when administered to a subject. That is, the variantanti-CD40 antibody will serve as a therapeutically active component inthe pharmaceutical composition in a manner similar to that observed forthe native antagonist antibody, for example 15B8 as expressed by thehybridoma cell line 15B8. Methods are available in the art fordetermining whether a variant anti-CD40 antibody retains the desiredbiological activity, and hence serves as a therapeutically activecomponent in the pharmaceutical composition. Biological activity ofantibody variants can be measured using assays specifically designed formeasuring activity of the native antagonist antibody, including assaysdescribed in the present invention.

Suitable biologically active variants of native or naturally occurringantagonist anti-CD40 antibodies can be fragments, analogues, andderivatives of that polypeptide. By “fragment” is intended a polypeptideconsisting of only a part of the intact polypeptide sequence andstructure, as noted elsewhere herein. By “analogue” is intended ananalogue of either the native polypeptide or of a fragment of the nativepolypeptide, where the analogue comprises a native polypeptide sequenceand structure having one or more amino acid substitutions, insertions,or deletions. By “derivative” is intended any suitable modification ofthe native polypeptide of interest, of a fragment of the nativepolypeptide, or of their respective analogues, such as glycosylation,phosphorylation, polymer conjugation (such as with polyethylene glycol),or other addition of foreign moieties, so long as the desired biologicalactivity of the native polypeptide is retained. Methods for makingpolypeptide fragments, analogues, and derivatives are generallyavailable in the art.

For example, amino acid sequence variants of an antagonist anti-CD40antibody can be prepared by mutations in the cloned DNA sequenceencoding the antibody of interest. Methods for mutagenesis andnucleotide sequence alterations are well known in the art. See, forexample, Walker and Gaastra, eds. (1983) Techniques in Molecular Biology(MacMillan Publishing Company, New York); Kunkel (1985) Proc. Natl.Acad. Sci. USA 82:488-492; Kunkel et al. (1987) Methods Enzymol.154:367-382; Sambrook et al. (1989) Molecular Cloning: A LaboratoryManual (Cold Spring Harbor, N.Y.); U.S. Pat. No. 4,873,192; and thereferences cited therein; herein incorporated by reference. Guidance asto appropriate amino acid substitutions that do not affect biologicalactivity of the polypeptide of interest may be found in the model ofDayhoff et al. (1978) in Atlas of Protein Sequence and Structure (Natl.Biomed. Res. Found., Washington, D.C.), herein incorporated byreference. Conservative substitutions, such as exchanging one amino acidwith another having similar properties, may be preferred. Examples ofconservative substitutions include, but are not limited to, Gly

Ala, Val

Ile

Leu, Asp

Glu, Lys

Arg, Asn

Gln, and Phe

Trp

Tyr.

In constructing variants of the antagonist anti-CD40 antibodypolypeptide of interest, modifications are made such that variantscontinue to possess the desired activity, i.e., similar binding affinityand having the following characteristics: 1) are capable of specificallybinding to a human CD40 antigen expressed on the surface of a humancell; 2) are free of significant agonist activity when bound to a CD40antigen on a normal human B cell; and, 3) exhibit antagonist activitywhen bound to a CD40 antigen on a malignant human B cell. Obviously, anymutations made in the DNA encoding the variant polypeptide must notplace the sequence out of reading frame and preferably will not createcomplementary regions that could produce secondary mRNA structure. SeeEP Patent Application Publication No. 75,444.

Biologically active variants of anti-CD40 antibodies will generally haveat least 70%, preferably at least 80%, more preferably about 90% to 95%or more, and most preferably about 98% or more amino acid sequenceidentity to the amino acid sequence of the reference polypeptidemolecule, which serves as the basis for comparison. A biologicallyactive variant of a reference antagonist anti-CD40 antibody having thespecificity and binding characteristics described herein may differ fromthe reference polypeptide by as few as 1-15 amino acids, as few as 1-10,such as 6-10, as few as 5, as few as 4, 3, 2, or even 1 amino acidresidue. By “sequence identity” is intended the same amino acid residuesare found within the variant polypeptide and the polypeptide moleculethat serves as a reference when a specified, contiguous segment of theamino acid sequence of the variant is aligned and compared to the aminoacid sequence of the reference molecule. The percentage sequenceidentity between two amino acid sequences is calculated by determiningthe number of positions at which the identical amino acid residue occursin both sequences to yield the number of matched positions, dividing thenumber of matched positions by the total number of positions in thesegment undergoing comparison to the reference molecule, and multiplyingthe result by 100 to yield the percentage of sequence identity.

For purposes of optimal alignment of the two sequences, the contiguoussegment of the amino acid sequence of the variants may have additionalamino acid residues or deleted amino acid residues with respect to theamino acid sequence of the reference molecule. The contiguous segmentused for comparison to the reference amino acid sequence will compriseat least twenty (20) contiguous amino acid residues, and may be 30, 40,50, 100, or more residues. Corrections for increased sequence identityassociated with inclusion of gaps in the variant's amino acid sequencecan be made by assigning gap penalties. Methods of sequence alignmentare well known in the art for both amino acid sequences and for thenucleotide sequences encoding amino acid sequences.

Thus, the determination of percent identity between any two sequencescan be accomplished using a mathematical algorithm. One preferred,non-limiting example of a mathematical algorithm utilized for thecomparison of sequences is the algorithm of Myers and Miller (1988)CABIOS 4:11-17. Such an algorithm is utilized in the ALIGN program(version 2.0), which is part of the GCG sequence alignment softwarepackage. A PAM120 weight residue table, a gap length penalty of 12, anda gap penalty of 4 can be used with the ALIGN program when comparingamino acid sequences. Another preferred, nonlimiting example of amathematical algorithm for use in comparing two sequences is thealgorithm of Karlin and Altschul (1990) Proc. Natl. Acad. Sci. USA87:2264, modified as in Karlin and Altschul (1993) Proc. Natl. Acad.Sci. USA 90:5873-5877. Such an algorithm is incorporated into the NBLASTand XBLAST programs of Altschul et al. (1990) J. Mol. Biol. 215:403.BLAST nucleotide searches can be performed with the NBLAST program,score=100, wordlength=12, to obtain nucleotide sequences homologous to anucleotide sequence encoding the polypeptide of interest. BLAST proteinsearches can be performed with the XBLAST program, score=50,wordlength=3, to obtain amino acid sequences homologous to thepolypeptide of interest. To obtain gapped alignments for comparisonpurposes, Gapped BLAST can be utilized as described in Altschul et al.(1997) Nucleic Acids Res. 25:3389. Alternatively, PSI-Blast can be usedto perform an iterated search that detects distant relationships betweenmolecules. See Altschul et al. (1997) supra. When utilizing BLAST,Gapped BLAST, and PSI-Blast programs, the default parameters of therespective programs (e.g., XBLAST and NBLAST) can be used. See thewebsite at ncbi.nlm.nih.gov. Also see the ALIGN program (Dayhoff (1978)in Atlas of Protein Sequence and Structure 5:Suppl. 3 (NationalBiomedical Research Foundation, Washington, D.C.) and programs in theWisconsin Sequence Analysis Package, Version 8 (available from GeneticsComputer Group, Madison, Wis.), for example, the GAP program, wheredefault parameters of the programs are utilized.

When considering percentage of amino acid sequence identity, some aminoacid residue positions may differ as a result of conservative amino acidsubstitutions, which do not affect properties of protein function. Inthese instances, percent sequence identity may be adjusted upwards toaccount for the similarity in conservatively substituted amino acids.Such adjustments are well known in the art. See, for example, Myers andMiller (1988) Computer Applic. Biol. Sci. 4:11-17.

The precise chemical structure of a polypeptide capable of specificallybinding CD40 and retaining antagonist activity, particularly when boundto CD40 antigen on malignant B cells, depends on a number of factors. Asionizable amino and carboxyl groups are present in the molecule, aparticular polypeptide may be obtained as an acidic or basic salt, or inneutral form. All such preparations that retain their biologicalactivity when placed in suitable environmental conditions are includedin the definition of antagonist anti-CD40 antibodies as used herein.Further, the primary amino acid sequence of the polypeptide may beaugmented by derivatization using sugar moieties (glycosylation) or byother supplementary molecules such as lipids, phosphate, acetyl groupsand the like. It may also be augmented by conjugation with saccharides.Certain aspects of such augmentation are accomplished throughpost-translational processing systems of the producing host; other suchmodifications may be introduced in vitro. In any event, suchmodifications are included in the definition of an anti-CD40 antibodyused herein so long as the antagonist properties of the anti-CD40antibody are not destroyed. It is expected that such modifications mayquantitatively or qualitatively affect the activity, either by enhancingor diminishing the activity of the polypeptide, in the various assays.Further, individual amino acid residues in the chain may be modified byoxidation, reduction, or other derivatization, and the polypeptide maybe cleaved to obtain fragments that retain activity. Such alterationsthat do not destroy antagonist activity do not remove the polypeptidesequence from the definition of anti-CD40 antibodies of interest as usedherein.

The art provides substantial guidance regarding the preparation and useof polypeptide variants. In preparing the anti-CD40 antibody variants,one of skill in the art can readily determine which modifications to thenative protein nucleotide or amino acid sequence will result in avariant that is suitable for use as a therapeutically active componentof a pharmaceutical composition used in the methods of the presentinvention.

Any pharmaceutical composition comprising an antagonist anti-CD40antibody as the therapeutically active component can be used in themethods of the invention. Thus liquid, lyophilized, or spray-driedcompositions comprising antagonist anti-CD40 antibodies or variantsthereof that are known in the art may be prepared as an aqueous ornonaqueous solution or suspension for subsequent administration to asubject in accordance with the methods of the invention. Each of thesecompositions will comprise anti-CD40 antibodies or variants thereof as atherapeutically or prophylactically active component. By“therapeutically or prophylactically active component” is intended theanti-CD40 antibody or variant thereof is specifically incorporated intothe composition to bring about a desired therapeutic or prophylacticresponse with regard to treatment, prevention, or diagnosis of a diseaseor condition within a subject when the pharmaceutical composition isadministered to that subject. Preferably the pharmaceutical compositionscomprise appropriate stabilizing agents, bulking agents, or both tominimize problems associated with loss of protein stability andbiological activity during preparation and storage.

Formulants may be added to pharmaceutical compositions comprising ananti-CD40antibody of the invention. These formulants may include, butare not limited to, oils, polymers, vitamins, carbohydrates, amineacids, salts, buffers, albumin, surfactants, or bulking agents.Preferably carbohydrates include sugar or sugar alcohols such as mono-,di-, or polysaccharides, or water soluble glucans. The saccharides orglucans can include fructose, glucose, mannose, sorbose, xylose,maltose, sucrose, dextran, pullulan, dextrin, α and β cyclodextrin,soluble starch, hydroxyethyl starch, and carboxymethylcellulose, ormixtures thereof. “Sugar alcohol” is defined as a C₄ to C₈ hydrocarbonhaving a hydroxyl group and includes galactitol, inositol, mannitol,xylitol, sorbitol, glycerol, and arabitol. These sugars or sugaralcohols may be used individually or in combination. The sugar or sugaralcohol concentration is between 1.0% and 7% w/v., more preferablybetween 2.0% and 6.0% w/v. Preferably amino acids include levorotary (L)forms of carnitine, arginine, and betaine; however, other amino acidsmay be added. Preferred polymers include polyvinylpyrrolidone (PVP) withan average molecular weight between 2,000 and 3,000, or polyethyleneglycol (PEG) with an average molecular weight between 3,000 and 5,000.Surfactants that can be added to the formulation are shown in EP Nos.270,799 and 268,110.

Additionally, antibodies can be chemically modified by covalentconjugation to a polymer to increase their circulating half-life, forexample. Preferred polymers, and methods to attach them to peptides, areshown in U.S. Pat. Nos. 4,766,106; 4,179,337; 4,495,285; and 4,609,546;which are all hereby incorporated by reference in their entireties.Preferred polymers are polyoxyethylated polyols and polyethylene glycol(PEG). PEG is soluble in water at room temperature and has the generalformula: R(O—CH₂—CH₂)_(n)O—R where R can be hydrogen, or a protectivegroup such as an alkyl or alkanol group. Preferably, the protectivegroup has between 1 and 8 carbons, more preferably it is methyl. Thesymbol n is a positive integer, preferably between 1 and 1,000, morepreferably between 2 and 500. The PEG has a preferred average molecularweight between 1,000 and 40,000, more preferably between 2,000 and20,000, most preferably between 3,000 and 12,000. Preferably, PEG has atleast one hydroxy group, more preferably it is a terminal hydroxy group.It is this hydroxy group which is preferably activated to react with afree amino group on the inhibitor. However, it will be understood thatthe type and amount of the reactive groups may be varied to achieve acovalently conjugated PEG/antibody of the present invention.

Water-soluble polyoxyethylated polyols are also useful in the presentinvention. They include polyoxyethylated sorbitol, polyoxyethylatedglucose, polyoxyethylated glycerol (POG), and the like. POG ispreferred. One reason is because the glycerol backbone ofpolyoxyethylated glycerol is the same backbone occurring naturally in,for example, animals and humans in mono-, di-, triglycerides. Therefore,this branching would not necessarily be seen as a foreign agent in thebody. The POG has a preferred molecular weight in the same range as PEG.The structure for POG is shown in Knauf et al. (1988) J. Bio. Chem.263:15064-15070, and a discussion of POG/IL-2 conjugates is found inU.S. Pat. No. 4,766,106, both of which are hereby incorporated byreference in their entireties.

Another drug delivery system for increasing circulatory half-life is theliposome. Methods of preparing liposome delivery systems are discussedin Gabizon et al. (1982) Cancer Research 42:4734; Cafiso (1981) BiochemBiophys Acta 649:129; and Szoka (1980) Ann. Rev. Biophys. Eng. 9:467.Other drug delivery systems are known in the art and are described in,e.g., Poznansky et al. (1980) Drug Delivery Systems (R. L. Juliano, ed.,Oxford, N.Y.) pp. 253-315; Poznansky (1984) Pharm Revs 36:277.

A further embodiment of the invention is the use of antagonist anti-CD40antibodies for diagnostic monitoring of protein levels in tissue as partof a clinical testing procedure, e.g., to determine the efficacy of agiven treatment regimen. Detection can be facilitated by coupling theantibody to a detectable substance. Examples of detectable substancesinclude various enzymes, prosthetic groups, fluorescent materials,luminescent materials, bioluminescent materials, and radioactivematerials. Examples of suitable enzymes include horseradish peroxidase,alkaline phosphatase, β-galactosidase, or acetylcholinesterase; examplesof suitable prosthetic group complexes include streptavidin/biotin andavidin/biotin; examples of suitable fluorescent materials includeumbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine,dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; anexample of a luminescent material includes luminol; examples ofbioluminescent materials include luciferase, luciferin, and aequorin;and examples of suitable radioactive material include ¹²⁵I, ¹³¹I, ³⁵S,or ³H.

The antagonist anti-CD40 antibodies can be used in combination withknown chemotherapeutics and cytokines for the treatment of diseasestates comprising malignant B cells. For example, the anti-CD40antibodies of the invention can be used in combination with cytokinessuch as interleukin-2 In another embodiment, the anti-CD40 antibodies ofthe invention can be used in combination with Rituximab (IDEC-C2B8;Rituxan®; IDEC Pharmaceuticals Corp., San Diego, Calif.). Rituximab is achimeric anti-CD20 monoclonal antibody containing human IgG1 and kappaconstant regions with murine variable regions isolated from a murineanti-CD20 monoclonal antibody, IDEC-2B8 (Reff et al. (1994) Blood83:435-445).

The anti-CD40 antibodies described herein can further be used to providereagents, e.g., labeled or labelable antibodies that can be used, forexample, to identify cells expressing CD40. This can be very useful indetermining the cell type of an unknown sample. Panels of monoclonalantibodies can be used to identify tissue by species and/or by organtype. In a similar fashion, these anti-CD40 antibodies can be used toscreen tissue culture cells for contamination (i.e., screen for thepresence of a mixture of CD40-expressing and non-CD40 expressing cellsin a culture).

The following examples are offered by way of illustration and not by wayof limitation.

EXPERIMENTAL

The antagonist anti-CD40 antibody used in the examples below is 15B8.15B8 is a human IgG₂ subtype anti-human CD40 monoclonal antibodygenerated by immunization of transgenic mice bearing the human IgG2heavy chain locus and the human K light chain locus (Xenomouse®,Abgenix). As shown by FACS analysis, 15B8 binds specifically to humanCD40 and cross-reacts with CD40 expressed on the peripheral blood Bcells from monkeys (cynomologus, rhesus and baboons) and chimpanzees.15B8 does not cross-react with CD40 from non-primate animal species, nordoes it bind to other members of the TNF receptor family as demonstratedby ELISA and FACS analysis. The binding affinity of 15B8 to human CD40is 3.1×10⁻⁹M as determined by Biacore™ assay.

Example 1 Effect of 15B8 on the CD40/CD40L Interaction In Vitro

A competitive binding assay was performed to determine if directcompetition for CD40 binding is a mechanism of the antagonist activityof 15B8.

A line of Chinese Hamster Ovary (CHO) cells containing the gene encodingCD40L and expressing CD40L on the cell surface was generated. TheCD40L-expressing CHO cells were incubated with purified CD40 before andafter incubation of CD40 with 15B8. Fluorescein isothiocyanate(FITC)-labeled anti-huIgG was added to the cells. FACS analysis wasperformed to detect 15B8 bound to the CHO cells via CD40. The binding of15B8 to CD40 inhibited the subsequent binding of CD40L to CD40. However,when CD40L and CD40 were incubated together prior to the addition of15B8, 15B8 was subsequently able to bind CD40. While not bound by anymechanism of action, this suggests that 15B8 does not compete directlywith CD40L for binding sites on CD40, and that the binding of 15B8 toCD40 possibly caused conformational changes in the CD40 molecule thatprevented the binding of CD40L to CD40. The putative structuralalteration of the CD40 molecule induced by 15B8 binding could alsodeliver a negative signal to the cell causing the antagonist effect.

Example 2 Pharmacologic Action of 15B8 in Lymphoma Cells from NHLPatients

To demonstrate the potential efficacy of 15B8 in a preclinical in vitromodel of non-Hodgkin's lymphoma (NHL), 15B8 was tested using malignant Bcells (NHL cells) obtained from NHL patients who were eitherRituximab-treated or naive. Rituximab (IDEC-C2B8; Rituxan®; IDECPharmaceuticals Corp., San Diego, Calif.) is an Anti-CD20 monoclonalantibody for the treatment of relapsed or refractory low-grade orfollicular NHL.

Since primary lymphoma cells do not proliferate in regular culturemedium and undergo apoptosis after a few days in culture, tumor cellswere co-cultured with irradiated CD40-ligand (CD40L) transfected feedercells (Arpin et al. (1995) Science 268:720-722) in the presence orabsence of the B-cell growth factor interleukin-4 (IL-4). Antibodies(agonist anti-CD40 MS81, antagonist anti-CD40 15B8, or isotype controlhuman IgG2 (huIgG2)) of indicated concentration (from 0.01 μg/ml to 10μg/ml) were then added to the culture. Following incubation at 37° C.for 48 hours, cultured cells were pulsed with ³H-thymidine for 18 hours.The cells were then harvested and analyzed for the amount of³H-thymidine incorporation (Schultze et al. (1995) Proc. Natl. Acad.Sci. USA 92:8200-8204). All sample conditions were in triplicate.

In these NHL cell primary culture assays, 15B8 alone or in combinationwith IL-4 did not stimulate NHL cells to proliferate in vitro. Incontrast, an agonist anti-CD40 MS81 induced NHL cell proliferation underthe same conditions. 15B8 showed statistically significant inhibition ofNHL cell proliferation stimulated by CD40L (P=0.05) and by CD40L plusIL-4 (P<0.05) in vitro. At 1-10 μg/ml or 0.1-10 μg/ml concentrationrange respectively, 15B8 showed a statistically significant dose-relatedinhibition of NHL cell-proliferation stimulated by CD40L or by CD40Lplus IL-4 (P<0.005) (data not shown).

There are two types of preclinical models that are currently used forevaluation of human antigen-specific monoclonal antibodies (Mabs) intherapeutic development for lymphomas. One model is the xenograft mousein vivo model, where the EBV-transformed lymphoma cell lines, such asDaudi (Burkitt lymphoma) or Raji (Burkitt lymphoma) cells, arexenografted into SCID/Nude mice. The defects of these models are thatthe results only reflect effects on the particular immortal cell line,which is derived from one EBV-transformed cell. It is known that Burkittlymphoma cells are lymphoblastoid cells (Ambinder et al. (1999) CancerTreat. Res. 99:27-45; Quintanilla-Martinez et al. (1998) Leuk. Lymphoma30:111-121; Klein (1996) Acta Microbiol. Immunol. Hung. 43:97-105) whilethe lymphoma cells from NHL patients are believed to be at the mature Bcell stage (Ghia et al. (2000) Adv. Cancer Res. 79:157-173). EBVtransformation of B cells results in changes of many components in theCD40 signaling pathway (Uchida et al. (1999) Science 286:300-303;Farrell et al. (1997) Biomed. Pharmacother. 51:258-267). In contrast toCD40 signaling in NHL cells and normal B cells, CD40 signaling leads togrowth arrest in EBV-transformed Burkitt lymphoma cell lines (Fukuda etal. (2000) Viral Immunol. 13:215-229; Baker et al. (1998) Blood92:2830-2843). Thus, the results of testing an antagonist anti-CD40 MAb(15B8) in the xenograft models will not be able to predict the responseto the antibody (15B8) by NHL patients.

The other model is the in vitro growth inhibition assay of lymphomacells from NHL patients, which was used above. The advantage is that theresults predicate the sensitivity of the lymphoma cells from NHLpatients to the agent (15B8) tested. However, the results are obtainedfrom in vitro study under defined conditions. A previous published studyreported that a rat anti-mouse CD40, which failed to induce ADCC and CDCin vitro, showed good efficacy in two syngeneic mouse B lymphoma models(BCL1 and A31) (Tutt et al. (1998) J. Immunol. 161:3176-3185). Theanti-tumor effect of the anti-mouse CD40 occurred slower in time than ananti-Id tested. One of the hypotheses was that the anti-mouse CD40operated by blocking critical growth signals that are dependent on theexpression of surface CD40, not direct signaling like anti-Id in themouse models tested. When tested, 15B8 did not bind to the Fcγ receptorsin vitro and failed to induce ADCC and CDC in vitro (data not shown)since it is of human IgG2 subtype. 15B8 has similar properties to therat anti-mouse CD40. These data support the hypothesis that 15B8 will bebeneficial to NHL patients, especially Rituxan®-resistant patients.

Example 3 Effect of 15B8 on Malignant B-Cell Proliferation In Vitro

To test if 15B8 provides the growth signal like CD40L in vitro, B cellsfrom tumor infiltrated lymph nodes (NHL cells) were obtained from oneantibody naïve, one Rituximab-sensitive and one Rituximab-resistant NHLpatient. The NHL cells were studied under four different cultureconditions: no added antibody (medium); addition of human isotypeantibody IgG2 (control; referred to as huIgG2); addition of anti-CD40antibody MS81 (agonistic antibody); and addition of 15B8. All antibodieswere tested at 1, 2, and 5 μg/ml in the presence or absence of IL-4. TheNHL cells from two patients were cultured as described above under thesame four conditions in the presence of IL-4 (2 ng/ml). B-cellproliferation was measured by ³H-thymidine incorporation as describedabove.

Anti-CD40 antibody 15B8, at concentrations of 1, 2, and 5 μg/ml, did notstimulate NHL cells to proliferate in either the absence or presence ofIL-4. In contrast, an agonistic anti-CD40 antibody (MS81), tested at thesame concentration, stimulated NHL-cell proliferation both in thepresence and absence of IL-4 in all patient samples. Representativeresults from one patient are shown in FIGS. 1 and 2. Results from theNHL cells from the two patients in the presence of IL-4 and threepatients in the absence of IL-4 were comparable. These results indicatethat 15B8 is not an agonist anti-CD40 antibody and does not stimulateproliferation of NHL cells from Rituximab-sensitive, naïve orRituximab-resistant NHL patients in vitro.

FACS analysis of the NHL cells was performed with either adirect-labeled 15B8-FITC or 15B8 plus anti-huIgG2-FITC to confirm thatCD40 is expressed on the surface the NHL cells tested and that 15B8binds to the NHL cells. The NHL cells from 2 Rituximab-sensitive and 4Rituximab-resistant patients (6 patients in total) were tested. NHLcells from all the patients expressed CD40 and bound 15B8. The 15B8binding-positive cell population in any given patient was about 66% to91%.

Example 4 15B8 Inhibits CD40L-Stimulated Proliferation of NHL Cells InVitro

To evaluate the ability of 15B8 to block the growth signal provided byCD40L in vitro, NHL cells from patients were cultured as described abovein suspension over CD40L-expressing feeder cells under four differentconditions: no added antibody (medium); addition of human isotypeantibody IgG2 (control); addition of anti-CD40 antibody MS81 (agonisticantibody); and addition of 15B8. All antibodies were added atconcentrations of 1, 2, and 5 μg/ml in the presence or the absence ofIL-4. The NHL cells from 1 antibody-naïve, 2 Rituximab-sensitive, and 5Rituximab-resistant patients (8 patients in total) were cultured underthe same four conditions as described above in the presence of IL-4 (2ng/ml). NHL cells from 3 Rituximab-sensitive and 4 Rituximab-resistantpatients (7 patients in total) were cultured under similar conditions inthe absence of IL-4. The NHL cell proliferation was measured by³H-thymidine incorporation.

Table 1 below shows the inhibitory effect of 15B8 on the proliferationof NHL cells from 2 Rituximab-sensitive (data from one patientreproducible in two separate experiments) and 4 Rituximab-resistantpatients (6 patients in total) stimulated by CD40L alone in vitro.Representative results from the cells of one patient (A) are shown inFIG. 3. 15B8 inhibited the proliferation by about 12-68% when comparedto the control in the 6 patients. The degree of inhibition by 15B8varied depending on patient samples and the dose level of 15B8.Statistical analysis of the data from 6 of the 7 patient samples testedshows that the inhibition of CD40L-stimulated NHL cell proliferation by15B8 is significant at 1 μg/ml (p=0.05). There is a statisticallysignificant dose response (p<0.005), as the inhibitory effect increaseswith increasing 15B8 dose.

TABLE 1 Effect of 15B8 MAb on CD40-L stimulation of proliferation of NHLpatient cells in the absence of IL-4.¹ Treatment Dose 15B8 Patient IDPatient Type² (μg/ml) % Inhibition³ A CR 1 56.61 2 58.99 5 63.16 A CR 161.96 2 60.41 5 64.75 10 60.29 B CR 1 None 2 None 5 None 10 12.11 D NR 152.22 2 61.63 5 68.04 10 68.17 E NR 1 13.07 2 22.34 5 31.04 10 31.87 FNR 1 24.51 2 27.43 5 38.71 10 47.35 G NR 1 11.12 2 22.41 5 30.61 1043.15 ¹NHL cells from patients were cultured with murine L-cellsexpressing human CD40L in the presence of medium, agonist anti-CD40(MS81), antagonist anti-CD40 (15B8), or huIgG2 isotype control in vitro.The proliferation of the NHL cells was measured by ³H-thymidineincorporation (data from one Rituximab-sensitive patient is not in thetable for the cpm of CD40L is <2000). ²Patient response to anti-CD20 Mabtherapy; CR, complete responder; NR, non-responder. ³15B8 % inhibition =100 − (15B8 cpm/huIgG2 cpm × 100); represents the mean of 3determinations.

Table 2 (below) shows the inhibitory effect of 15B8 on proliferation ofNHL cells from 1 antibody-naïve, 2 Rituximab-sensitive (data from bothpatient samples were repeated twice reproducibly), and 5Rituximab-resistant patients (8 patients in total) stimulated by bothCD40L and IL-4 in vitro. At 1 μg/ml level, 15B8 significantly (p<0.05)inhibited the CD40L and IL-4-mediated proliferation of the NHL cells.The degree of inhibition ranged from 18-69% at high dose (5 or 10 μg/ml)in samples from all 8 patients in vitro. There was a statisticallysignificant dose response of this inhibitory effect by 15B8 (p<0.005) ata 15B8 concentration range of 0.01-10 μg/ml. FIG. 4 shows onerepresentative dose response curve. These in vitro results suggest thattreatment with 15B8 may block the CD40-mediated growth signal for NHLcells in patients.

TABLE 2 Effect of 15B8 Mab on CD40-L stimulation of NHL patient cells inthe presence of IL-4.¹ Treatment Dose 15B8 Patient ID Patient Type²(μg/ml) % Inhibition³ A CR 1 34.39 2 30.54 5 36.42 A CR 0.01 0.44 0.0423.32 0.2 29.54 1 35.38 5 46.12 10 48.63 C CR 1 34.91 2 40.89 5 56.34 1069.21 C CR 1 None 2 16.79 5 21.64 10 12.63 D NR 1 1.95 2 6.43 5 20.95 1026.31 E NR 1 1.91 2 2.74 5 28.36 10 28.26 E NR 1 None 2 11.76 5 27.54 1034.07 G NR 1 39.38 2 32.74 5 36.48 10 37.78 H NR 1 None 2 None 5 7.81 1018.47 I Naive 0.01 None 0.04 13.16 0.2 15.64 1 16.20 5 21.53 10 24.51¹NHL cells from patients were cultured with murine L-cells expressinghuman CD40L in the presence of IL-4 (human interleukin-4) at 2 ng/mlunder conditions described in Table 1. ²Patient response to anti-CD20Mab therapy; CR, complete responder; NR, non-responder; Naïve,untreated. ³% inhibition compared to huIgG2. 15B8 % inhibition = 100 −(15B8 cpm/huIgG2 cpm × 100).

Example 5 15B8 Does Not Activate Human Peripheral Blood B Cells and DoesNot Cause PBMC Proliferation In Vitro in Human, Chimpanzee, and Marmoset

To determine if it is an agonist or antagonist anti-CD40, 15B8 wastested in several in vitro assays described below using cells fromhumans and five different primate species, including chimpanzee (chimp),marmoset, cynomologus monkey, rhesus monkey, and baboon.

TABLE 3 Stimulation of PBMC/B-cell proliferation in human, chimp, andmarmoset by 15B8 antibody.¹ Cell Number of Dose huIgG2, CD40L, Fold15B8, Fold Species Source Samples (μg/ml) Base Increase³ Increase² HumanB 2 5 1 70.58/36.33 1.77/4.37 2 1 1 70.58/36.33 3.1/5.4 2 0.2 170.58/36.33 1.16/4.63 Human PBMC 5 5 1 9.36-91.60 0.49-2.28 15 1 19.36-91.60 0.35-2.38 12 0.2 1 9.36-91.60 0.41-3.74 Marmoset PBMC 3 5 129.24-90.3  2.05-7.2  Monkey 5 1 1 7.99-90.3  1.35-5.79 Chimp PBMC 1 5 110.15 2.46 5 1 1 5.12-9.2  0.66-5.2  ¹B cells/PBMCs were cultured invitro in the presence of CD40L, 15B8, or huIgG2 isotype control.²Results of the cell proliferation are reported as the ratio of³H-thymidine incorporation for 15B8 to huIgG2 control. Data from somesamples are not included in the table for the CPM induced by CD40L(positive control) <2000. ³The fold-increase for CD40L shown in thetable is the ratio of the CD40L cpm to the cpm of huIgG2 at 5 μg/ml.

Upon B-cell activation, a number of cell surface proteins areup-regulated (Denton et al. (1998) Pediatr. Transplant. 2:6-15; Evans etal. (2000) J. Immunol. 164:688-697; Noelle (1998) Agents Actions Suppl.49:17-22; Lederman et al. (1996) Curr. Opin. Hematol. 3:77-86). Toconfirm 15B8 does not activate human B cells and does not induce anagonist signal when bound to CD40, its ability to up-regulate B cellactivation markers was tested by FACS analysis using purified humanPBMC. There was no up-regulation in the expression of activation markerssuch as CD25, CD69, CD86, HLA-DR, and ICAM-1 (CD54) in 15B8 treatedhuman B cells (Table 4). The level of these markers was similar whencells were treated with either 15B8 or huIgG2 control (Table 4). Incontrast, CD69 was consistently up-regulated by CD40L in PBMC samplesfrom 3 healthy volunteers tested.

TABLE 4 Effect of 15B8 on up-regulation of B-cell activation markers invitro by FACS. Cell Incubation Number of Species Source Time SubjectsCD54 CD69 HLA-DR CD25 CD80 CD86 Human CD20 4 h-24 h 3 — — — — N/A — fromPBMC Chimp CD20 4 h-24 h 3 N/A — N/A N/A N/A N/A from PBMC ¹“—” means noup-regulation. ²“N/A” means not measured or not successful.

Additional consequences of B cell activation are up-regulation ofsurface FasL and apoptosis (Revy et al. (1998) Eur. J. Immunol.28:3648-3654; Carey et al. (2000) Immunol. Rev. 176:105-115; Ju et al.(1999) Int. Rev. Immunol. 18:485-513; Baumgarth (2000) Immunol. Rev.176:171-180). To confirm 15B8 is not an agonistic anti-CD40 antibody,its ability to induce FasL expression and apoptosis of human B cells wasalso tested. Annexin V staining on the cell surface can be used as anearly apoptosis marker (Ju et al. (1999) Int. Rev. Immunol. 18:485-513).Human B cells were purified from peripheral blood and incubated with15B8. FACS analysis was used to detect cells with positive staining ofAnnexin V and anti-FasL. There was no significant difference on thesurface staining by the two reagents between cells incubated with 15B8or the isotype control (huIgG2) antibody (data not shown). This resultshows that 15B8 does not induce apoptosis of human B cells in vitro.These data provide further evidence that 15B8 is not an agonistanti-CD40 antibody for human B cells.

15B8 cross-reacts with CD40 expressed on the surface of CD20 positivePBMCs from primates. To test if 15B8 can activate CD40 on B cells fromother primate species such as chimpanzees and marmosets, the sameproliferation assays were carried out using freshly isolated chimp andmarmoset PBMC from 15 chimps and 5 marmosets. Similar to the resultswith the human PBMC, 15B8 did not stimulate the proliferation in vitroof PBMCs from 6 chimps and 5 marmosets at 1 and 5 μg/ml concentration(Table 3 above). 15B8 also did not up-regulate the expression ofactivation marker, CD69, in the three chimp-PBMC samples tested (Table4). 15B8 did not show any effect on FasL expression and apoptosis inchimp PBMCs similar to human PBMC controls after 24 and 48 hoursstimulation in vitro in all samples from 6 chimps tested (data notshown).

Cross-linking 15B8 by a secondary antibody fixed to plastic surface didnot increase its potency to stimulate B-cell proliferation (data notshown). When tested using PBMCs from humans and chimps in thiscross-linking assay, 15B8 did not stimulate proliferation of the cells.This observation indicates a reduced risk of 15B8 being stimulative(i.e., agonistic or having agonist activity) for B-cell proliferation incase of induction of anti-15B8 (HAHA) or Fc binding to other Fc receptorexpressing cells when administered in vivo.

In summary, 15B8 does not initiate an activation signal in human Bcells/PBMCs nor in chimp/marmoset PBMCs in vitro. Therefore, 15B8 is notan agonist anti-CD40 antibody in human, chimps, and marmosets.

Example 6 15B8 is an Antagonist Anti-CD40 Antibody in Humans,Chimpanzees, and Marmosets In Vitro

To determine if 15B8 is an antagonist anti-CD40, its ability to inhibitCD40-CD40L interaction was tested in a CD40L-mediated human B-cellproliferation assay (Kwekkeboom et al. (1993) Immunology 79:439-444). Atransfected CHO cell line expressing human CD40L was used to stimulatethe proliferation of purified human peripheral blood B cells or PBMCs.Human B cells from 10 healthy volunteers and human PBMCs from 3 healthyvolunteers were tested. In all the samples tested, 15B8 suppressedCD40L-expressing CHO cells mediated-proliferation by 42-88% atconcentration range from 0.2-5 μg/ml (Table 5). FIG. 5 shows arepresentative dose-response curve using cells from 3 individuals. Theno-effect dose of 15B8 is 0.008 μg/ml and reaches saturating dose at 0.2μg/ml (FIG. 5). This observation indicates that 15B8, as an antagonistanti-CD40 antibody, can inhibit the growth signals in human B cells andPBMCs provided by cell surface-expressed CD40L.

TABLE 5 Inhibition of CD40L-inducted-proliferation of PBMC/B cell with15B8 antibody.¹ Cell Number of Dose CD40L HuIgG2, % of 15B8, % ofSpecies Source Samples (μg/ml) (Base) Inhibition Inhibition² Human B 7 5100 (−27)-14% 45-85% 9 1 100 (−93)-11% 42-87% 6 0.2 100  (−20)-(−6)%44-82% Human PBMC 1 5 100 13% 45% 2 1 100     3-32% 76-88% Marmoset PBMC3 1 100     1-35% 68-84% Monkey Chimpanzee PBMC 3 1 100  (−3)-21% 55-73%¹B cells/PBMCs were cultured in vitro with CD40L-expressing CHO cells inthe presence of 15B8 or huIgG2 control. CD40L-transfected CHO cells werefixed with formaldehyde before the experiments. The proliferation ofcells was measured by ³H-thymidine incorporation. ²“15B8 % inhibition” =100 − (15B8 cpm/CD40L cpm × 100). Data from some samples are not in thetable for proliferation inducted by CD40L (positive control) is <5-fold.

Additional assays were carried out using freshly isolated PBMCs from 9chimps and 3 marmosets. As with the human PBMCs, 15B8 was able toinhibit the proliferation of chimp and marmoset PBMCs stimulated byCD40L-expressing-CHO cells at 1 μg/ml concentration level (Table 5above). The inhibition by 15B8 was approximately 55-73% and 68-84% inPBMC samples from 3 chimps and 3 marmosets respectively (Table 5 above).

Activated B cells undergo a number of biological responses such asproliferation and antibody production. The activation of B cells by Tcell-dependent antigens involves CD4⁺ T-helper (Th) cells. This T cellhelper process is mediated by a concerted effort of the interaction ofCD40 on the B cells with the CD40L on the Th cells surface together withthe interactions of other co-stimulatory factors and cytokines (Dentonet al. (1998) Pediatr. Transplant. 2:6-15; Evans et al. (2000) J.Immunol. 164:688-697; Noelle (1998) Agents Actions Suppl. 49:17-22;Lederman et al. (1996) Curr. Opin. Hematol. 3:77-86; Mackey et al.(1998) J. Leukoc. Biol. 63:418-428). To test if 15B8 can block T-helpercell mediated B cell antibody production, purified human peripheralblood B cells were cultured in the presence of purified irradiated Tcells activated with anti-CD3 antibody. An ELISA assay was used tomeasure the level of IgM production. 15B8 reduced IgM production byabout 30% in this assay (data not shown). Therefore, 15B8 can reduceT-cell-mediated B-cell immunoglobulin production.

In summary, 15B8 inhibits CD40L-induced B-cell/PBMC proliferation inhuman, chimp and marmoset, and inhibits T-cell induced antibodyproduction by purified human B cells in vitro. These data demonstratethat 15B8 is an antagonist anti-CD40 antibody in human B cells and PBMCsfrom chimps and marmosets in vitro.

Example 7 15B8 is an Agonist Anti-Monkey (Cynomologus, Rhesus, andBaboon) CD40 Antibody In Vitro

FACS analysis demonstrates that 15B8 binds to CD40 expressed on thesurface of B cells from peripheral blood of monkeys (rhesus,cynomologus, and baboon). The effect of 15B8 on freshly isolatedcynomologus monkey PBMC was tested in the same proliferation assaydescribed above for human and chimps (Coligan et al. (1998) CurrentProtocols in Immunology 13:12; Kwekkeboom et al. (1993) Immunology79:439-444). In contrast to human PBMC, 15B8 was found to stimulatecynomologus monkey PBMC to proliferate in vitro as measured by ³Hmethyl-thymidine incorporation (Table 6 below). At 1 μg/ml level, 15B8stimulated the proliferation of the PBMCs by 6-fold to 129.7-foldcompare to the huIgG2 control in the twenty-two samples from 17 monkeystested (samples from 5 monkeys were tested twice) (Table 6 below). At 5μg/ml level, the proliferation stimulated by 15B8 is 14-fold to 24-foldin four samples from 2 monkeys and about 1.25-fold or 1.85-fold in twosamples from 2 monkeys (Table 6). This suggests that, at concentrationlevel of 5 μg/ml, 15B8 may be at the limit of over-saturating dose forits proliferation-stimulatory effect on PBMCs from cynomologus monkey.Further FACS analysis of B cells for activation status by surfacemarkers indicated that 15B8 induces CD69, CD86, and HLA-DR up-regulationon monkey B cells (Table 7). These data suggest that 15B8 is an agonistantibody to CD40 expressed on peripheral blood B cells from cynomologusmonkeys in vitro.

To confirm that this agonistic effect of 15B8 is not cynomologus-monkeyspecific, the same assays were performed using PBMCs from rhesus monkeysand baboons. Similar results to that obtained from cells of cynomologusmonkeys were observed as shown in Table 6. 15B8 stimulated proliferationof PBMCs from rhesus monkeys and baboons in vitro (Table 6). The agonistactivity of 15B8 is shown using the PBMCs from 5 rhesus monkeys and 3baboons (Table 6).

TABLE 6 Proliferation of PBMCs from human, cynomologus and rhesusmonkeys, and baboons stimulated by 15B8.¹ Cell Number of Dose hulgG2,CD40L, Fold 15B8, Fold Species Source Samples (ug/ml) Base Increase³Increase² Human PBMC 5 5 1 9.36-91.60 0.49-2.28 15 1 1 9.36-91.600.35-2.38 12 0.2 1 9.36-91.60 0.41-3.74 Rhesus PBMC 5 1 1 12.71-89.67 27.34-50.9  Monkey Cyno PBMC 6 5 1 14.57-124.01  1.25-24.53 Monkey 22 11  5.15-167.73  6.13-129.74 3 0.2 1 77.01-124.01  0.9-67.56 Baboon PBMC3 1 1  5.19-175.07  3.32-113.28 ¹PBMCs were cultured in vitro in thepresence of CD40L, 15B8, or huIgG2 control. ²The proliferation resultsare reported as the ratio of ³H-thymidine incorporation for 15B8 tohuIgG2 control. Data from some samples are not in the table for the CPMinduced by CD40L (positive control) <2000. ³The fold-increase for CD40Lshown in the table is the ratio of the CD40L cpm to the cpm of huIgG2 at5 μg/ml. CD40L transfected CHO cells were fixed with formaldehyde beforethe experiments.

TABLE 7 Effect of 15B8 on upregulation of B-cell activation markers invitro by FACS analysis. Cell incubation Number of Species source timesubject CD54 CD69 HLA-DR CD25 CD80 CD86 Human CD20 4 h-24 h  3 — — — —N/A — from PBMC Cyno CD20/19 4 h-3 day 2 N/A 1/2 up 1.1 up — — 1/1 upMonkey from (day 3) (day 3) PBMC ¹“—” means no up-regulation. ²“N/A”means not measured or not successful. ³Only cells from one cynomologusmonkey was analyzed by FACS on day 3 because of limited cell number.

Example 8 15B8 is an Agonist Anti-CD40 Antibody In Vivo in CynomologusMonkeys

15B8 can stimulate proliferation and up-regulation of cell surfaceactivation markers in PBMCs from cynomologus monkeys in vitro. Todetermine if 15B8 is an agonist anti-CD40 antibody in these monkeys invivo, a study was performed to examine the biodistribution of 15B8 andthe fate of affected peripheral B cells (i.e., extravasation, apoptosis,activation status, or complement lysis) [Biodistribution of 15B8.72Antibodies following Intravenous Administration to Non-Naïve Male andFemale Cynomologus Monkeys (SNBL.218.3, SNBL USA)].

Cynomologus monkeys (1 female and 2 males) received a single intravenousadministration of 3 mg/kg 15B8. The following parameters were monitored:clinical signs, food consumption, body weight, pharmacokinetics, serumcomplement (CHSO), flow cytometry for B cells (including apoptotic Bcells), T cells, and monocytes. B-cell CD40 receptor saturation with15B8 was also measured. Animals were necropsied 24 hours after receivingthe single dose of 15B8, and standard organs were weighed. Pre-studysurgical biopsies of spleen and axillary lymph nodes were taken to serveas baseline controls. At necropsy, lymphoid and non-lymphoid tissueswere sampled for histopathology and immunohistochemistry. Tissues wereimmunostained with antibodies against CD3, CD40, CD20, CD27, and CD38antigens. Preliminary results of the study are discussed below.

All animals survived to the scheduled necropsy and there were no effectson food consumption, body weight, CHSO levels, nor on peripheral bloodT-cell or monocyte counts. There were no changes in organ weights.Microscopic examination of the spleen showed moderate diffuse follicularhyperplasia with necrosis and/or neutrophilic infiltrates in thegerminal centers of all 15B8-treated animals. Examination of mesentericand inguinal lymph nodes revealed mild follicular hyperplasia in 2 outof 3 animals. No treatment related microscopic effects were seen inother tissues (liver, skin, brain, thyroid, lung, bone marrow, adrenalgland, and kidney).

Immunostaining with CD20, CD27, CD40, and CD86 antibodies revealedincreases in these markers in splenic and lymph node follicles, whichcorrelated with the follicular hyperplasia seen in these same tissues.Increased staining of CD20 and CD40 were limited to the spleen and lymphnode while there was some additional staining of hepatic tissue withCD27 and of hepatic Kupffer cells and inflammatory cells by CD86. CD86staining was also increased in thymic medullary cells and adrenalinterstitial leukocytes. There were no changes in the immunostaining ofCD3 in 15B8-treated animals as compared to controls.

These findings indicate that a single dose of 3 mg/kg of 15B8administered to cynomologus monkey can cause proliferation of lymphoidfollicles and/or redistribution of B cells from the peripheral blood inspleen and lymph nodes within a 24-hour period. Antibodies to CD20,CD27, CD40, and CD86 recognize antigens expressed on B cells and/oractivated B cells, along with recognition of other cell types. Increasednumbers of cells expressing these antigens were seen in the spleen andlymph nodes of treated animals, which suggests an increase in the numberof activated CD20+ B cells. This study suggests that 15B8 is an agonistanti-CD40 antibody in cynomologus monkey in vivo. The results obtainedin vivo and in vivo are consistent in cynomologus monkeys.

Example 9 Effect of 15B8 on Peripheral B Cells in Chimpanzees

Two groups of 3 male chimpanzees received either 0.03 mg/kg or 3 mg/kg15B8 by intravenous administration. Serum 15B8 concentrations andperipheral B cell numbers were monitored immediately after 15B8administration and through day 29 post-dose. The results of theexperiment are shown in FIG. 6. After administration of 15B8 at 3 mg/kg,serum 15B8 concentrations declined in a triphasic pattern involving ashort distribution phase, a log-linear elimination phase, and anon-linear elimination phase. The non-linear elimination phasepredominated at concentrations below approximately 10 μg/ml. Thehalf-life during the log linear phase was approximately four days.Peripheral B cell numbers decreased immediately after 15B8administration and recovered within 3-4 weeks. 15B8 was detected inserum, bound to surface CD40 receptors on circulating B cells. Theextent of binding appeared to remain relatively unchanged from Day 2through 8 post-dose and declined subsequently through Day 29 post-dose.

After administration of 15B8 at 0.03 mg/kg, B cells appeared to declineslightly by 30 minutes but returned to pre-dose values within 4 hours.Serum 15B8 concentrations were below the level of detection at 30minutes after dosing.

Example 10 ELISA Assay for Immunoglobulin Quantification

The concentrations of human IgM and IgG are estimated by ELISA assays.96-well ELISA plates are coated with 4 μg/ml anti-human IgG mAb or with1.2 μg/ml anti-human IgM mAb in 0.05 M carbonate buffer (pH=9.6) for 16hours at 4° C. Plates are washed three times with PBS-0.05% Tween-20(PBS-Tween) and saturated with BSA for one hour. After two washes, theplates are incubated for one hour at 37° C. with different dilutions ofthe test samples. After three washes, bound Ig is detected by incubationfor one hour at 37° C. with 1 μg/ml peroxidase labeled mouse anti-humanIgG mAb or mouse anti-human IgM mAb. Plates are washed four times andbound peroxidase activity is revealed by the addition ofo-phenylenediamine as a substrate.

CONCLUSIONS

Summary of the In Vitro Assays

The results suggest that 15B8 is an agonistic anti-CD40 antibody incynomologus and rhesus monkeys and baboons, and an antagonistic antibodyin humans, chimpanzees, and marmosets. The experiments that have beencompleted are summarized in the tables below.

TABLE 8 Assays measuring agonistic activity. Species Tested AssayMethodology (+ or − Agonistic Activity) Effect of 15B8 on B cellCompared ³H-thymidine Human (−) proliferation incorporation of purifiedB cells from the peripheral blood in presence of 15B8 with incorporationin presence of CD40L or an agonistic antibody 626.1 Effect of 15B8 onPBMC Compared ³H-thymidine Human (−) proliferation incorporation ofPBMCs in Chimpanzee (−) presence of 15B8 with Cynomologus monkey (+)incorporation in presence of Rhesus monkey (+) CD40L or the isotypecontrol Baboon (+) Marmoset (−) Effect of 15B8 on Measured upregulationin the Human (−) upregulation of B-cell expression of B-cell activationChimpanzee (−) activation markers markers in PBMCs stimulated byCynomologus monkey (+) 15B8 or its isotype control using Rhesus monkey(+) FACS analysis; compared effect Baboon (+) of 15B8 with that ofisotype Marmoset (−) control Effect on PBMC proliferation Compared³H-thymidine Human (−) of 15B8 cross-linked to a incorporation inpresence of Chimpanzee (−) secondary antibody fixed to a secondAb-crosslinked 15B8 with plastic surface incorporation in presence ofCD40L 15B8 alone or the isotype control Effect of 15B8 on Measuredupregulation in the Human (−) upregulation of FasL and expression ofFasL and apoptosis Chimps (−) apoptosis by FACS detection of B cellsCynomologus Monkey (−/+) with positive staining of anti- FasL andAnnexin V (marker for apoptosis) by the stimulation of CD40L, 15B8, andthe isotype control.

TABLE 9 Assays measuring antagonistic activity. Species Tested (+ or −Antagonistic Assay Methodology Activity) Inhibition by 15B8 ofStimulation of B-cell proliferation Human (+) CD40L-mediated B-cell byCD40L-expressing CHO cells Marmoset (+) proliferation was measured by³H-thymidine Chimps (+) incorporation. Compared ³H- thymidineincorporation in presence of 15B8 with that in presence of isotypecontrol Inhibition by 15B8 of T-helper- B cells were cultured with Human(+) cell-mediated B-cell antibody purified irradiated T cells productionactivated with anti-CD3 antibody in the presence of 15B8. The level ofB-cell IgM production was assessed by ELISA.

15B8 is an anti-human CD40 specific monoclonal antibody with human IgG₂subtype and with cross-reactivity to CD40 from non-human primates only.Through extensive in vitro testing, 15B8 was shown to be an antagonistanti-CD40 to the CD40 expressed on human B cells, PBMCs from human,chimp, and marmoset. However, 15B8 was shown to have agonist activitywhen bound to the CD40 expressed on PBMCs from monkeys (cynomologus,rhesus, and baboon) in vitro. This agonist activity of 15B8 wasconfirmed in vivo in cynomologus monkeys. When tested in primary cultureof lymphoma cells from Rituxan®-sensitive and resistant NHL patients,15B8 has no agonist activity in the presence or absence of IL-4. 15B8can also inhibit CD40L-stimulated growth of the lymphoma cells from thesimilar group of patients under both conditions. 15B8 has the potentialto modify B-cell malignancies, such as non-Hodgkin's lymphoma (NHL),where the CD40/CD40L pathway may play a role in the pathogenesis of thediseases.

All publications and patent applications mentioned in the specificationare indicative of the level of those skilled in the art to which thisinvention pertains. All publications and patent applications are hereinincorporated by reference to the same extent as if each individualpublication or patent application was specifically and individuallyindicated to be incorporated by reference.

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

1. A method for treating a patient with a disease comprising malignant B cells, said method comprising administering at least one therapeutically effective dose of a human anti-CD40 monoclonal antibody, an antigen-binding fragment of said antibody, or a conjugated form of said antibody or said antigen-binding fragment to said patient, wherein said anti-CD40 antibody or antigen-binding fragment, or conjugated form thereof, exhibits antagonist activity when said antibody or antigen-binding fragment, or conjugated form thereof, binds a CD40 antigen on a malignant human B cell, wherein said human anti-CD40 monoclonal antibody is selected from the group consisting of: a) a monoclonal antibody comprising a light chain variable region having the amino acid sequence set forth in SEQ ID NO:2; b) a monoclonal antibody comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO:4; c) a monoclonal antibody having a light chain variable region having the amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO:1; d) a monoclonal antibody having a heavy chain variable region having the amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO:3; and e) a monoclonal antibody that comprises the complementarity determining regions (CDRs) of the monoclonal antibody produced by the hybridoma cell line 15B8 (Patent Deposit Designation PTA-3814); wherein said antigen-binding fragment of said antibody retains the capability of specifically binding to human CD40, and wherein said therapeutically effective dose of said human anti-CD40 monoclonal antibody or antigen-binding fragment, or conjugated form thereof, is in the range from about 0.01 mg/kg to about 40 mg/kg, whereby said disease is treated.
 2. The method of claim 1, wherein said malignant B cells are selected from the group consisting of B-cell lymphoma cells, non-Hodgkin's lymphoma cells, high-grade B-cell lymphoma cells, intermediate-grade B-cell lymphoma cells, low-grade B-cell lymphoma cells, B-cell acute lymphoblastic leukemia cells, multiple myeloma cells, chronic lymphocytic leukemia cells, myeloblastic leukemia cells, and Hodgkin's disease cells.
 3. The method of claim 1, wherein said antigen-binding fragment is selected from the group consisting of a Fab fragment, an F(ab′)₂ fragment, and Fv fragment, and a single-chain Fv fragment.
 4. The method of claim 1, wherein said treating comprises administration of multiple therapeutically effective doses of said human anti-CD40 monoclonal antibody or antigen-binding fragment, or conjugated form thereof, to said patient.
 5. The method of claim 4, wherein said malignant B cells are selected from the group consisting of B-cell lymphoma cells, non-Hodgkin's lymphoma cells, high-grade B-cell lymphoma cells, intermediate-grade B-cell lymphoma cells, low-grade B-cell lymphoma cells, B-cell acute lymphoblastic leukemia cells, multiple myeloma cells, chronic lymphocytic leukemia cells, myeloblastic leukemia cells, and Hodgkin's disease cells.
 6. The method of claim 5, wherein said antigen-binding fragment is selected from the group consisting of a Fab fragment, an F(ab′)₂ fragment, and Fv fragment, and a single-chain Fv fragment. 